Methods and nucleic acids for determining the prognosis of a cancer subject

ABSTRACT

The invention provides methods, nucleic acids and kits for determining the prognosis of a subject having cancer. The invention discloses genomic sequences the methylation patterns of which have utility for the improved detection of said disorder, thereby enabling the improved diagnosis and treatment of patients.

FIELD OF THE INVENTION

The present invention relates to genomic DNA markers useful indetermining the prognosis of a cancer subject, determining medicaltreatment for a cancer subject, determining if a tumor from a cancersubject indicates that the tumor is aggressive or has metastaticpotential or indicates a reduced survival time for the subject,detecting an aggressive form of cancer in a subject, selecting a cancersubject for cancer treatment, or determining tumor load or cancer burdenin a subject. Particular embodiments provide methods, nucleic acids,nucleic acid arrays and kits useful for determining the prognosis of asubject having cancer.

BACKGROUND

Methods for determining the prognosis, and thus methods and agents fordetermining treatment, of a cancer patient include determining thestaging of the tumor based on various criteria. Often this determinationincludes invasive procedures to observe histological changes in tissuemorphology and the level of invasion of the tumor into neighboringtissue and metastasis.

In particular, colorectal cancer is the second most frequent cancer inEurope and in the the US (412,900 and 150,000 individuals in 2006,respectively). In 75% of cases disease is removed by surgery. However,there is recurrence in 30-40% of stage II-III colorectal cancers, mostwithin 3-5 years of initial diagnosis. Moreover, only 16-66% of patientsare symptomatic at diagnosis of recurrence and of these tumors only1.7-7% are resectable. Thus, 93-98.3% of recurrent cases are identifiedpast the time where resection is sufficient to remove all of the tumoror tumor cells. See Fakih, M. G. MD, CEA Monitoring in ColorectalCancer, What You Should Know, Volume 20: Number 6: 2006.

Current practice guidelines for post-resection surveillance for Stage IIand greater tumors include monitoring CEA (Carcinoembryonic antigen)every 3-6 months for 2 years then every 6 months for a total of 5 years,and/or colonoscopy after 1 year, optionally repeated every second year.For colorectal Stage I and II patients who are positive for CEA beforesurgery, only 3% to 32% of patients can be monitored by CEA-basedmonitoring, leaving 68-97% of Stage I & II patients who cannot bemonitored at all with CEA. Furthermore, CEA sensitivity depends on thesite of recurrence such that only a portion of the 3-32% of patients whocan be monitored can benefit.

Currently, the only valid prognostic marker in predicting the outcome ofcolorectal cancer (CRC) patients is the Tumor-Node-Metastais (TNM)staging system. The parameters of this system are generally qualitativeand are not informative for further differentiating risk in standardrisk patients, who constitute the majority of stage II colon cancer.Approximately 30% of patients with colon cancer have a stage II disease.Current National Comprehensive Cancer Network (NCCN) guidelines do notrecommend the routine use of adjuvant chemotherapy for all patients withstage II colon cancer but rather consider adjuvant treatment in thesetting of high recurrence risk. The five-year survival rate for theoverall stage II patient population has been estimated to be 75-80%.Despite these relatively high cure rates with surgery alone, in asignificant proportion of stage II patients cancer will recur. Theidentification of markers that distinguish those patients at low riskfrom those at higher risk of disease recurrence, would be helpful toidentify those patients who would be candidates for adjuvantchemotherapy. Biomarkers in stage II colon cancer to date have beenlimited to clinical diagnosis, but not use in prognosis or clinicaloutcome.

Several proteins and genetic markers have been described in an attemptto improve prognostic information and to predict the benefit fromsystemic treatment. Unlike other types of cancer, with the exception ofKRAS mutation, none of the studied markers has entered into the clinicalmanagement of colorectal cancer so far.

CpG island methylation: Aberrant methylation of CpG islands has beenshown to lead to the transcriptional silencing of certain genes thathave been previously linked to the pathogenesis of various cellproliferative disorders, including cancer. CpG islands are sequencesthat are rich in CpG dinucleotides and can usually be found in the 5′region of approximately 50% of all human genes. Methylation of thecytosines in these islands leads to the loss of gene expression and hasbeen reported in the inactivation of the X chromosome and genomicimprinting.

DNA methylation and disease prognosis: DNA methylation has been shown tobe associated with patient prognosis in a number of publications such asEP 1692316 and WO 2007/085497.

There is a need for a better means to determine a patient's prognosis,clinical outcome, tumor load, cancer burden, and/or inclusion in atreatment group, at any point starting at initial diagnosis andcontinuing during the course of treatment, including the ability todetermine the status of relapse, remission, or recurrence, usingminimally invasion testing techniques.

SUMMARY OF THE INVENTION

The invention provides a method for determining the prognosis of acancer subject, comprising the steps of: measuring the pre-treatmentlevel of methylated genomic DNA of a gene, or a fragment thereof, in abiological sample obtained from the subject; measuring thepost-treatment level of methylated genomic DNA of the gene or a fragmentthereof, in a biological sample obtained from the subject, whereby anincreased or equivalent amount of the methylated genomic DNA or fragmentin the post-treatment sample compared to the pre-treatment sampleindicates additional cancer treatment for the subject. Within anembodiment, an increased amount of the methylated genomic DNA orfragment in the post-treatment sample compared to the pre-treatmentsample indicates that the cancer is aggressive or has metastaticpotential or reduced survival time for the subject. In a preferredembodiment the method of the invention provides a method for determiningthe prognosis of a cancer subject, comprising the steps of: a) measuringthe pre-treatment level of methylated genomic DNA of a gene, or afragment thereof, in a biological sample obtained from the subject; b)measuring the post-treatment level of methylated genomic DNA of the geneor a fragment thereof, in a biological sample obtained from the subject;and c) comparing the measured post-treatment level and the measuredpre-treatment level of methylated DNA, whereby an increased orequivalent amount of the methylated genomic DNA or fragment in thepost-treatment sample as compared to the pre-treatment sample indicatesa bad prognosis and, thus, a need for additional cancer treatment forthe subject. In a preferred embodiment of the method, the methodcomprises steps c) and d) as follows: c) comparing the measuredpost-treatment level and the measured pre-treatment level of methylatedDNA and d) determining the prognosis of a cancer subject based on theresult of the comparison of step c), whereby an increased or equivalentamount of the methylated genomic DNA or fragment in the post-treatmentsample as compared to the pre-treatment sample indicates a bad prognosisand, thus, a need for additional cancer treatment for the subject.

Stable or even increased levels of the methylated gcnomic DNA,preferably, indicate that the chosen treatment failed to remove thecancer cells discharging the methylated DNA fragment or that theirnumber increased despite of treatment, i.e. the cancer grew further. Incontrast to this, decreased levels of the methylated genomic DNA,preferably, indicate that the number of cancer cells decreased, i.e.that the treatment was successful in reducing tumor load of the patient.In particular, a decrease to levels which are below the level ofdetection, indicates that all cancer cells may have been eradicated fromthe patient, i.e. a cure of the cancer. Typically, a cancer whichresponds poorly to treatment is considered aggressive.

If the applied cancer treatment is localized treatment, a decrease ofthe level of the methylated genomic DNA to a level below the limit ofdetection, preferably indicates a cure of the cancer. It will beunderstood by the person skilled in the art that—depending on theoutcome of clinical studies—other threshold levels for defining a “cure”of a patient may be defined. The establishment of such threshold levelscan be achieved by statistical methods conventional in the field of(medical) statistics.

However, if the level of the methylated genomic DNA measured afterlocalized treatment is above the level of detection, this, preferably,indicates that localized treatment was insufficient to achieve acomplete cure. This is typically the case if the cancer already spreadbeyond the area affected by the localized treatment. Therefore, even inthe case of a decrease of the level of the methylated genomic DNA, thecontinued presence of detectable levels of the methylated genomic DNAindicates a poor prognosis because a cancer which spreads beyond itssite of origin is, typically, much more difficult to treat.

The selection of further treatment of a cancer patient depends onhis/her prognosis. If the prognosis is good, subsequent treatment doesnot need to be as aggressive as in cases with a bad prognosis. As theprognosis of the patient is an important parameter for the selection offurther treatment of a cancer patient, the invention provides a methodfor determining medical treatment for a cancer subject, comprising thesteps of: measuring the pre-treatment level of methylated genomic DNA ofa gene, or a fragment thereof, in a biological sample obtained from thesubject; measuring the post-treatment level of methylated genomic DNA ofthe gene or a fragment thereof, in a biological sample obtained from thesubject, whereby an increased or equivalent amount of the methylatedgenomic DNA or fragment in the post-treatment sample compared to thepre-treatment sample indicates additional cancer treatment for thesubject. In a preferred embodiment the invention also provides a methodfor determining which kind of medical treatment is suitable for a cancersubject, comprising the steps of: a) measuring the pre-treatment levelof methylated genomic DNA of a gene, or a fragment thereof, in abiological sample obtained from the subject; b) measuring thepost-treatment level of methylated genomic DNA of the gene or a fragmentthereof, in a biological sample obtained from the subject; and c)comparing the measured post-treatment level and the measuredpre-treatment level of methylated DNA, whereby an increased orequivalent amount of the methylated genomic DNA or fragment in thepost-treatment sample compared to the pre-treatment sample indicatesadditional cancer treatment for the subject. In a preferred embodimentof the method, the method comprises steps c) and d) as follows: c)comparing the measured post-treatment level and the measuredpre-treatment level of methylated DNA and d) determining based on theresult of the comparison of step c) which kind of medical treatment issuitable for a cancer subject, whereby an increased or equivalent amountof the methylated genomic DNA or fragment in the post-treatment samplecompared to the pre-treatment sample indicates additional cancertreatment for the subject.

Preferably, a post-treatment level of the methylated genomic DNA whichdecreased below the level of detection indicates that no further medicaltreatment is required. In these cases, a monitoring of the patient forrelapses may be sufficient. However, if the post-treatment level of themethylated genomic DNA does not decrease or even increases, additionalmedical treatment may be necessary. As an increasing level of themethylated genomic DNA indicates a failure of the treatment, thissituation, preferably, indicates the need to switch to a different kindof treatment.

It will be understood by the person skilled in the art that the choiceof a suitable treatment of a cancer patient cannot be not exclusivelybased on the result of a single laboratory test. This decision is,preferably, based on medical judgement of the patient's condition. Saidjudgement, preferably includes results of conventional diagnosticmethods such as imaging methods as well as the general stat of health ofthe particular patient in addition to the results gained by applying themethod of the present invention.

The invention provides a method for determining if a tumor from a cancersubject indicates that the tumor is aggressive or has metastaticpotential or indicates a reduced survival time for the subjectcomprising: measuring the pre-treatment level of methylated genomic DNAof a gene, or a fragment thereof, in a biological sample obtained fromthe subject; and measuring the post-treatment level of methylatedgenomic DNA of the gene or a fragment thereof, in a biological sampleobtained from the subject, whereby an increased or equivalent amount ofthe methylated genomic DNA or fragment in the post-treatment samplecompared to the pre-treatment sample indicates that the cancer isaggressive or has metastatic potential or indicates a reduced survivaltime for the subject. In a preferred embodiment the invention provides amethod for determining if a tumor from a cancer subject indicates thatthe tumor is aggressive or has metastatic potential or indicates areduced survival time for the subject comprising: a) measuring thepre-treatment level of methylated genomic DNA of a gene, or a fragmentthereof, in a biological sample obtained from the subject; b) measuringthe post-treatment level of methylated genomic DNA of the gene or afragment thereof, in a biological sample obtained from the subject; andc) comparing the measured post-treatment level and the measuredpre-treatment level of methylated DNA, whereby an increased orequivalent amount of the methylated genomic DNA or fragment in thepost-treatment sample compared to the pre-treatment sample indicatesthat the tumor is aggressive or has metastatic potential or indicates areduced survival time for the subject. In a preferred embodiment of themethod, the method comprises steps c) and d) as follows: c) comparingthe measured post-treatment level and the measured pre-treatment levelof methylated DNA and d) determining based on the result of thecomparison of step c) if a tumor from a cancer subject indicates thatthe tumor is aggressive or has metastatic potential or indicates areduced survival time for the subject, whereby an increased orequivalent amount of the methylated genomic DNA or fragment in thepost-treatment sample compared to the pre-treatment sample indicatesthat the tumor is aggressive or has metastatic potential or indicates areduced survival time for the subject.

Moreover, the invention provides a method for determining if a tumorfrom a cancer subject is aggressive and/or has metastatic potentialcomprising the steps of a) measuring the pre-treatment level ofmethylated genomic DNA of a gene, or a fragment thereof, in a biologicalsample obtained from the subject; b) measuring the post-treatment levelof methylated genomic DNA of the gene or a fragment thereof, in abiological sample obtained from the subject; c) comparing the measuredpost-treatment level and the measured pre-treatment level of methylatedDNA, whereby an increased or equivalent amount of the methylated genomicDNA or fragment in the post-treatment sample compared to thepre-treatment sample indicates that the tumor is aggressive and/or hasmetastatic potential. In a preferred embodiment of the method, themethod comprises steps c) and d) as follows: c) comparing the measuredpost-treatment level and the measured pre-treatment level of methylatedDNA and d) determining based on the result of the comparison of step c)if a tumor from a cancer subject is aggressive and/or has metastaticpotential, whereby an increased or equivalent amount of the methylatedgenomic DNA or fragment in the post-treatment sample compared to thepre-treatment sample indicates that the tumor is aggressive and/or hasmetastatic potential.

The invention provides a method for detecting an aggressive form ofcancer in a subject, comprising a) measuring the pre-treatment level ofmethylated genomic DNA of a gene, or a fragment thereof, in a biologicalsample obtained from the subject; b) measuring the post-treatment levelof methylated genomic DNA of the gene or a fragment thereof, in abiological sample obtained from the subject, whereby an increased amountof the methylated genomic DNA or fragment in the post-treatment samplecompared to the pre-treatment sample indicates that the cancer is anaggressive form. In a preferred embodiment the invention provides amethod for detecting an aggressive form of cancer in a subject,comprising a) measuring the pre-treatment level of methylated genomicDNA of a gene, or a fragment thereof, in a biological sample obtainedfrom the subject; b) measuring the post-treatment level of methylatedgenomic DNA of the gene or a fragment thereof, in a biological sampleobtained from the subject; and c) comparing the measured post-treatmentlevel and the measured pre-treatment level of methylated DNA, whereby anincreased amount of the methylated genomic DNA or fragment in thepost-treatment sample compared to the pre-treatment sample indicatesthat the cancer is an aggressive form. In a preferred embodiment of themethod, the method comprises steps c) and d) as follows: c) comparingthe measured post-treatment level and the measured pre-treatment levelof methylated DNA and d) detecting based on the result of the comparisonof step c) an aggressive form of cancer in a subject, whereby anincreased amount of the methylated genomic DNA or fragment in thepost-treatment sample compared to the pre-treatment sample indicatesthat the cancer is an aggressive form.

The invention provides a method for selecting a cancer subject forcancer treatment comprising: measuring the pre-treatment level ofmethylated genomic DNA of a gene, or a fragment thereof, in a biologicalsample obtained from the subject; and measuring the post-treatment levelof methylated genomic DNA of the gene or a fragment thereof, in abiological sample obtained from the subject, whereby an increase in theamount of methylated genomic DNA of the gene in the post-treatmentsample compared to the pre-treatment sample indicates additional cancertreatment. In a preferred embodiment the invention provides a method forselecting a cancer subject for additional cancer treatment comprising:measuring the pre-treatment level of methylated genomic DNA of a gene,or a fragment thereof, in a biological sample obtained from the subject;and measuring the post-treatment level of methylated genomic DNA of thegene or a fragment thereof, in a biological sample obtained from thesubject; comparing the measured post-treatment level and the measuredpre-treatment level of methylated DNA, whereby an increase in the amountof methylated genomic DNA of the gene or the fragment thereof or anequivalent amount of said DNA or the fragment thereof in thepost-treatment sample compared to the pre-treatment sample indicates theneed for additional cancer treatment. In a preferred embodiment of themethod, the method comprises steps c) and d) as follows: c) comparingthe measured post-treatment level and the measured pre-treatment levelof methylated DNA and d) selecting based on the result of the comparisonof step c) a cancer subject for additional cancer treatment, whereby anincrease in the amount of methylated genomic DNA of the gene or thefragment thereof or an equivalent amount of said DNA or the fragmentthereof in the post-treatment sample compared to the pre-treatmentsample indicates the need for additional cancer treatment.

Consequently, the present invention provides a method for determiningthe success of a treatment against cancer in a subject comprising thesteps of a) measuring the pre-treatment level of methylated genomic DNAof a gene, or a fragment thereof, in a biological sample obtained fromthe subject; and b) measuring the post-treatment level of methylatedgenomic DNA of the gene or a fragment thereof, in a biological sampleobtained from the subject; c) comparing the measured post-treatmentlevel and the measured pre-treatment level of methylated DNA, whereby(i) an decrease in the amount of methylated genomic DNA of the gene orthe fragment thereof in the post-treatment sample compared to thepre-treatment sample indicates that the treatment was successful and(ii) an increase in the amount of methylated genomic DNA of the gene orthe fragment thereof or an equivalent amount of said DNA or the fragmentthereof in the post-treatment sample compared to the pre-treatmentsample indicates that the treatment was not successful. In a preferredembodiment of the method, the method comprises steps c) and d) asfollows: c) comparing the measured post-treatment level and the measuredpre-treatment level of methylated DNA and d) determining based on theresult of the comparison of step c) the success of a treatment againstcancer in a subject, whereby (i) an decrease in the amount of methylatedgenomic DNA of the gene or the fragment thereof in the post-treatmentsample compared to the pre-treatment sample indicates that the treatmentwas successful and (ii) an increase in the amount of methylated genomicDNA of the gene or the fragment thereof or an equivalent amount of saidDNA or the fragment thereof in the post-treatment sample compared to thepre-treatment sample indicates that the treatment was not successful.

Preferably, a treatment which was “successful” achieved at least one ofthe following effects: remission of the cancer, increase of the time torecurrence of the cancer, increase of the time to tumor progression,alleviation of the symptoms of the cancer, reduction of tumor mass anddecrease of the number tumors. More preferably, a “successfultreatment”, characterized by a cure of the cancer, i.e. the completeeradication detectable and non-detectable tumor cells. A preferredindicator of the cure of the cancer is a recurrence free survival of thepatient for at least 5 years or, more preferably, at least 10 years.

A treatment which was “not successful”, preferably, failed to achieveany of the aims described above.

The invention provides a method for determining tumor load or cancerburden in a subject comprising: measuring the pre-treatment level ofmethylated genomic DNA of a gene, or a fragment thereof, in a biologicalsample obtained from the subject; and measuring the post-treatment levelof methylated genomic DNA of the gene or a fragment thereof, in abiological sample obtained from the subject; whereby an increase in theamount of methylated gcnomic DNA of the gene in the post-treatmentsample compared to the pre-treatment sample indicates that the subjecthas increased or equivalent tumor load or cancer burden or that thetumor load or cancer burden has not been diminished by the treatment. Ina preferred embodiment the invention provides a method for determiningthe development of tumor load or cancer burden in a subject comprising:a) measuring the pre-treatment level of methylated genomic DNA of agene, or a fragment thereof, in a biological sample obtained from thesubject; b) measuring the post-treatment level of methylated genomic DNAof the gene or a fragment thereof, in a biological sample obtained fromthe subject; and c) comparing the measured post-treatment level with themeasured pre-treatment level of methylated DNA, whereby an increase inthe amount of methylated genomic DNA of the gene or the fragment thereofor an equivalent amount of said DNA or the fragment thereof in thepost-treatment sample compared to the pre-treatment sample indicatesthat the subject has increased or equivalent tumor load or cancer burdenor that the tumor load or cancer burden has not been diminished by thetreatment. In a preferred embodiment of the method, the method comprisessteps c) and d) as follows: c) comparing the measured post-treatmentlevel and the measured pre-treatment level of methylated DNA and d)determining based on the result of the comparison of step c) thedevelopment of tumor load or cancer burden in a subject, whereby anincrease in the amount of methylated genomic DNA of the gene or thefragment thereof or an equivalent amount of said DNA or the fragmentthereof in the post-treatment sample compared to the pre-treatmentsample indicates that the subject has increased or equivalent tumor loador cancer burden or that the tumor load or cancer burden has not beendiminished by the treatment.

The invention provides a method for determining tumor load or cancerburden in a subject comprising comparing the post-treatment level ofmethylated genomic DNA of a gene or a fragment thereof, in a biologicalsample obtained from the subject with the pre-treatment level ofmethylated genomic DNA of the gene or fragment, whereby an increase inthe amount of methylated genomic DNA of the gene in the post-treatmentsample compared to the pre-treatment sample indicates that the subjecthas increased or equivalent tumor load or cancer burden or that thetumor load or cancer burden has not been diminished by the treatment. Ina preferred embodiment of the method, the method comprises steps c) andd) as follows: c) comparing the measured post-treatment level and themeasured pre-treatment level of methylated DNA and d) determining basedon the result of the comparison of step c) tumor load or cancer burdenin a subject, whereby an increase in the amount of methylated genomicDNA of the gene in the post-treatment sample compared to thepre-treatment sample indicates that the subject has increased orequivalent tumor load or cancer burden or that the tumor load or cancerburden has not been diminished by the treatment.

The level of methylated DNA of the genes of the present invention isgenerally useful as a marker for properties of a cancer such asaggressiveness or tumor load. A comparison of the levels of methylatedDNA taken at different points in time, therefore, indicatesindependently of ongoing treatment how the properties of the cancerdevelop over time.

For this reason, the present invention provides a method for monitoringa property of a cancer selected from the group consisting of tumor load,cancer burden, aggressiveness of a cancer and the prognosis of a cancersubject comprising the steps of a) measuring the level of methylatedgenomic DNA of a gene, or a fragment thereof, in a first biologicalsample obtained from a subject suffering from cancer; b) measuring thelevel of methylated genomic DNA of the gene or a fragment thereof in afurther biological sample obtained from the subject; and c) comparingthe measured levels of methylated DNA in the further sample and thefirst sample, wherein an increased level of methylated DNA of the geneor the fragment thereof in the further sample indicates that the tumorload, tumor burden or the aggressiveness of the cancer increased or theprognosis of the patient worsened and (ii) a decreased level ofmethylated DNA of the gene or the fragment thereof in the further sampleindicates that the tumor load, tumor burden or the aggressiveness of thecancer decreased or the prognosis of the patient improved.

In a preferred embodiment of the method, the method comprises steps c)and d) as follows: c) comparing the measured levels of methylated DNA inthe further sample and the first sample and d) determining based on theresult of the comparison of step c) whether the tumor load, tumor burdenor the aggressiveness of the cancer increased or decreased or theprognosis of the patient worsened or improved.

The first and second sample can be taken any time provided that thesecond sample is taken after the first sample. Preferably, the secondsample is taken at least 1 month, at least 2 months, at least 3 months,at least 6 months, at least 9 months or at least 12 months after thefirst sample.

The above-described method for monitoring a property of the cancer isespecially suitable for monitoring a patient whose cancer has alreadybeen treated before for recurrence and/or progression of the cancer.Thus, in a particularly preferred embodiment of the present invention,the patient is a cancer patient whose treatment apparently cured thecancer. The determination of methylation of the genes of the presentinvention in at least 2 samples taken at different points in time aftertreatment can be used to detect a recurrence of the cancer. It is ageneral problem in the field of cancer therapy that a treatment may beapparently effective, i.e. it decreases the tumor burden of the patientbelow the level which is detectable with the available diagnosticmethods, in particular imaging methods. Nevertheless, a few cancer cellsmay remain despite apparently successful treatment. These cells mayproliferate and cause a relapse of the cancer even years after anapparently successful treatment. Therefore, a follow-up of treatedpatients for some period of time after treatment is good medicalpractice in order to detect a relapse as early as possible. As themethod of the present invention is both sensitive (Septin 9, inparticular, may be used to detect early stages of colon carcinoma), easyto perform and non-invasive, it is particularly suited to monitortreated cancer patients during follow-up.

Within an aspect of the methods of the invention, the gene is SEPTIN9(SEQ ID NO:1) or RASSF2a (SEQ ID NO:16).

Within another aspect of the methods of the invention, the gene isSEPTIN9 (SEQ ID NO:1).

Within another aspect of the methods of the invention, the gene isRASSF2A (SEQ ID NO:16).

In a further preferred embodiment of the invention the above-describedmethods are based on the measurement of the level of methylated DNA ofboth SEPTIN9 and RASSF2A.

Within another aspect of the method of the invention, the cancer isselected from the group consisting of: colon cancer; and colorectalcancer. Within an embodiment, the stage of the cancer is Stage Icolorectal cancer. Within another embodiment, the stage of the cancer isStage II colorectal cancer. Within another embodiment, the cancer isStage III colorectal cancer. Within another embodiment, the cancer isStage IV colorectal cancer.

Within another aspect of the methods of the invention, the treatment isselected from the group consisting of: surgery or resection;immunotherapy; radiation; chemotherapy; therapy targeting solid tumors;therapy targeting soft-tissue tumors; and therapy targeting blood cells.

Within another aspect of the methods of the invention, the treatment islocalized to the region of cancer/tumor in the subject. Within anotheraspect of the methods of the invention, the treatment is not localizedto the region of cancer/tumor in the subject.

The term “localized treatment” preferably refers to surgical resectionof the tumor and/or radiation therapy. The term “not localizedtreatment” is equivalent to systemic treatment and, preferably, refersto chemotherapy and/or immunotherapy.

Within another aspect of the methods of the invention, the biologicalsample is selected from the group consisting of: tissue, blood, stool,urine, and lung lavage fluid, breast, prostate, colon, rectum, or acombination of these tissues. Within an embodiment, the sample is serumor plasma. The use of serum or plasma is preferred.

Within another aspect of the methods of the invention, methylatedgenomic DNA or fragment thereof is measured quantitatively or measuredquantitatively in part. Within another aspect of the methods of theinvention, methylated genomic DNA or fragment is measured qualitativelyor measured qualitatively in part. Within another aspect of the methodsof the invention, methylated genomic DNA or fragment is measuredquantitatively in part and qualitatively in part or semiquantitativley.

Within another aspect of the methods of the invention, measuring themethylated genomic DNA or fragment comprises contacting genomic DNA fromthe biological sample with at least one reagent, or series of reagentsthat distinguishes between methylated and non-methylated CpGdinucleotides within at least one target region of the genomic DNA,wherein the target region comprises, or hybridizes under stringentconditions to a sequence of at least 9, at least 16 or at least 25contiguous nucleotides of SEQ ID NOs: 1, 2, 3 or 16 wherein saidcontiguous nucleotides comprise at least one CpG dinucleotide sequence.Within an embodiment, contacting the genomic DNA, or the fragmentthereof in b), comprises use of a reagent selected from the groupcomprising of bisulfite, hydrogen sulfite, disulfite, and combinationsthereof.

Within another aspect of the methods of the invention comprise: a)extracting or otherwise isolating the genomic DNA or fragment thereoffrom the biological samples; b) treating the extracted or isolatedgenomic DNA or a fragment thereof with one or more reagents to convertcytosine bases that are unmethylated in the 5-position thereof to uracilor to another base that is detectably dissimilar to cytosine in terms ofhybridization properties; c) contacting the treated genomic DNA ortreated fragment, with an amplification enzyme and at least one primercomprising, a contiguous sequence of at least 9, at least 10, at least11, at least 12, at least 13, at least 14, at least 15, at least 16, atleast 17, at least 19, at least 20, at least 25, or at least 50nucleotides that is complementary to, or hybridizes under moderatelystringent or stringent conditions to a the treated sequence or to acomplement thereof, wherein the treated genomic DNA or the fragmentthereof is either amplified to produce at least one amplificate, or isnot amplified; and d) determining, based on a presence, absence oramount of, or on a property of said amplificate, the methylation stateor level of at least one CpG dinucleotide of the gene, or an average, ora value reflecting an average methylation state or level of a pluralityof CpG dinucleotides of the gene. The treated genomic DNA referred toabove is preferably selected from the group consisting of SEQ ID NO: 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19 and 20.

Within another aspect of the methods of the invention comprises a)extracting or otherwise isolating the genomic DNA or fragment thereoffrom the biological samples; b) digesting the extracted or isolatedgenomic DNA or a fragment thereof with one or more methylation sensitiverestriction enzymes; c) contacting the DNA restriction enzyme digest ofb), with an amplification enzyme and at least two primers suitable forthe amplification of a sequence comprising at least one CpG dinucleotideof the gene; and d) determining, based on a presence, absence or classof an amplificate the methylation state or level of at least one CpGdinucleotide of the gene.

Further information on preferred methods for measuring the level of amethylated genomic DNA can be found further below in the application. Inan especially preferred embodiment of the present invention the methodfor measurement of methylation levels of genomic DNA is MethyLight™,HeavyMethl™ or methylation specific PCR.

Within another aspect the invention provides a methylated genomicSEPTIN9 nucleic acid or a fragment comprising at at least 9, at least10, at least 11, at least 12, at least 13, at least 14, at least 15, atleast 16, at least 17, at least 19, at least 20, at least 25, or atleast 50 contiguous nucleotides of the nucleic acid and sequencescomplementary thereto for use in the determination of prognosis of acancer subject. Another embodiment of the present invention provides amethylated genomic RASSF2A nucleic acid or a fragment comprising atleast 9, at least 16, at least 25, or at least 50 contiguous nucleotidesof the nucleic acid and sequences complementary thereto for use in thedetermination of prognosis of a cancer subject. Within an embodiment thesubject has is colorectal cancer.

Within another aspect the invention provides the use of methylatedgenomic SEPTIN9 nucleic acid or a fragment comprising at least 9, atleast 16, at least 25, or at least 50 contiguous nucleotides of thenucleic acid and sequences complementary thereto for determining theprognosis of a cancer subject. Within an embodiment, the subject hascolorectal cancer.

Within another aspect the invention provides a bisulfite treated genomicSEPTIN9 or RASSF2A DNA nucleic acid comprising at at least 9, at least10, at least 11, at least 12, at least 13, at least 14, at least 15, atleast 16, at least 17, at least 19, at least 20, at least 25, or atleast 50 contiguous nucleotides, or a complement thereto for use indetermining the prognosis of a cancer subject. Preferably, the sequenceof the bisulfite treated SEPTIN9 or RASSF2A DNA is defined by SEQ ID NO:4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19 or 20. Within anembodiment, the contiguous base sequence comprises at least one CpG, TpGor CpA dinucleotide sequence.

Within another aspect the invention provides a kit for determining theprognosis of a cancer subject, determining medical treatment for acancer subject, for determining if a tumor from a cancer subjectindicates that the tumor is aggressive or has metastatic potential orindicates a reduced survival time for the subject, for detecting anaggressive form of cancer in a subject, for selecting a cancer subjectfor cancer treatment, or for determining tumor load or cancer burden ina subject comprising: a) a plurality of oligonucleotides orpolynucleotides able to hybridise under stringent or moderatelystringent conditions to the transcription products of the gene ormethylated genomic DNA; and b) means to detect the hybridisation. Withinan embodiment, the gene or methylated genomic DNA is SEPTIN9. Within anembodiment, the gene or methylated genomic DNA is RASSF2A.

Within another aspect the invention provides a kit for determining theprognosis of a cancer subject, determining medical treatment for acancer subject, for determining if a tumor from a cancer subjectindicates that the tumor is aggressive or has metastatic potential orindicates a reduced survival time for the subject, for detecting anaggressive form of cancer in a subject, for selecting a cancer subjectfor cancer treatment, or for determining tumor load or cancer burden ina subject comprising: (a) a bisulfite reagent; (b) at least one set ofoligonucleotides containing two oligonucleotides whose sequences in eachcase are identical , are complementary, or hybridize under stringent orhighly stringent conditions to a at at least 9, at least 10, at least11, at least 12, at least 13, at least 14, at least 15, at least 16, atleast 17, at least 19, at least 20, at least 25, or at least 50nucleotide long segment of a SEPTIN9 sequence or of a RASSF2A gene.

Within other aspects the invention provides the use of the methodsdescribed herein, the nucleic acids as described herein and/or a kit asdescribed herein for determining the prognosis of a cancer subject.

The present invention provides a method for determining the prognosis ofa subject having cancer, in a subject comprising determining theexpression levels of at least one gene or genomic sequence wherein thegenomic sequence is methylated in cancers and unmethylated innon-cancerous tissues. Methylation of the genomic DNA encoding a geneor, in particular, methylation of its promoter region decreases theexpression of said gene. Consequently, methylation of the gene inquestion gives a similar diagnostic information as its underexpression.Thus, the level or amount of methylation/or expression of the gene in abiological sample isolated from said subject is indicative of theprognosis of said subject. Various aspects of the present inventionprovide genetic markers, whereby expression analysis of said markerenables the determination of the prognosis of a subject having cancer.In one embodiment said expression level is determined by detecting thepresence, absence or level of mRNA transcribed from said gene. In afurther embodiment said expression level is determined by detecting thepresence, absence or level of a polypeptide encoded by said gene orsequence thereof.

The present invention provides a method for determining the prognosis ofa subject having colorectal cancer (CRC) or colon cancer, in a subjectcomprising determining the DNA Methylation levels of Septin 9 (Septin9)or of RASSF2A in plasma isolated from said subject wherein afterresection of the primary tumor the methylation status is indicative ofthe prognosis of said subject. In an embodiment the resection iscurative.

The examples described herein showed that the Septin9 biomarkerdecreases in approximately 73% of the investigated CRC Stage II and onlyin 20% of the Stage III patients after resection of the primary tumor.The presence of Septin9 in CRC patients after treatment with curativeintention can be used an early prognostic indicator of diseaserecurrence. The fact that Septin9 is still detectable after resection ofthe primary tumor, indicates a high risk of the presence of tumor cells(e.g. micro metastasis) that are still in the body of the patient andwhich can be sensitive detected by Septin9.

In further embodiments said expression is determined by detecting thepresence, absence or amount of CpG methylation within said gene, andtherefrom deducing the prognosis of said subject having cancer. Saidmethod comprises the following steps: i) contacting genomic

DNA isolated from a biological sample obtained from the subject with atleast one reagent, or series of reagents that distinguishes betweenmethylated and non-methylated CpG dinucleotides within at least onetarget region of the genomic DNA, wherein the nucleotide sequence ofsaid target region comprises at least one CpG dinucleotide sequence ofat least one gene or genomic sequence of this group of genes and ii)determining the prognosis of a subject having cancer. Preferably thetarget region comprises, or hybridizes under stringent conditions to asequence of at least 16, at least 25 or at least 50 contiguousnucleotides.

Said use of the gene may be enabled by means of any analysis of theexpression of the gene, by means of mRNA expression analysis or proteinexpression analysis. In an embodiment the determination of the prognosisof a subject having cancer, is enabled by means of analysis of themethylation status of at least one gene or genomic sequence that ismethylated in cancer tissue but unmethylated in non-cancerous tissue,including isoforms, fragments, promoter or regulatory elements, andantisense versions thereof.

The invention provides a method for the analysis of biological samplesfor features associated with the progression of cancer, the methodcharacterized in that the nucleic acid, or a fragment thereof iscontacted with a reagent or series of reagents capable of distinguishingbetween methylated and non methylated CpG dinucleotides within thegenomic sequence. In an embodiment, the gene is SEPTIN9 or RASSF2A.

Preferably, the sequence of SEPTIN9 is defined by SEQ ID NO: 1, 2 or 3.More preferably, the sequence of SEPTIN9 is defined by SEQ ID NO: 2 or3. The sequence of RASSF2A is, preferably, defined by SEQ ID NO: 16.

In a preferred embodiment of the present invention the methylationstatus of the promotor region of SEPTIN9 and/or RASSF2A is determined.In a more preferred embodiment, the methylation state of at least onecytosine comprised by the genomic sequence as defined by SEQ TD NO: 32and/or 34 is determined. In an even more preferred embodiment of theinvention, the methylation status of at least one cytosine selected fromthe group consisting of the cytosines in positions 21, 28, 30, 37 and 39of SEQ ID NO: 32 and positions 25, 29, 46, 52, 58, 70, 74, 79 and 89 ofSEQ ID NO: 34 is determined. In the most preferred embodiment, themethylation status of all aforementioned cytosine positions in SEQ IDNO: 32 and/or 34 is determined.

The present invention provides a method for ascertaining epigeneticparameters of genomic DNA associated with the development of cancer.

The source of the test sample is a tissue, or body fluid, such as, forexample, tissues and body fluids selected from the group consisting oftissue, blood, plasma, serum, urine, lung lavage fluid, stool, lung,breast, colon, rectum, intestine and combinations thereof.

Specifically, the present invention provides a method for determiningthe prognosis of a subject having cancer suitable for use in aprognostic tool, comprising: obtaining a biological sample comprisinggenomic nucleic acid(s); contacting the nucleic acid(s), or a fragmentthereof, with a reagent or a plurality of reagents sufficient fordistinguishing between methylated and non methylated CpG dinucleotidesequences within a target sequence of the subject nucleic acid, whereinthe target sequence comprises, or hybridises under stringent conditionsto, a sequence comprising at least 16, at least 25 or at least 50contiguous nucleotides of the gene said contiguous nucleotidescomprising at least one CpG dinucleotide sequence; and determining,based at least in part on said distinguishing, the methylation state ofat least one target CpG dinucleotide sequence, or an average, or a valuereflecting an average methylation state of a plurality of target CpGdinucleotide sequences.

In distinguishing between methylated and non methylated CpG dinucleotidesequences within the target sequence comprises methylationstate-dependent conversion or non-conversion of at least one such CpGdinucleotide sequence to the corresponding converted or non-converteddinucleotide sequence within a sequence selected from the groupconsisting of bisulfite converted sense and antisense strands of thegenes and contiguous regions thereof corresponding to the targetsequence.

Additional embodiments provide a method for the determination of theprognosis of a subject having cancer comprising: obtaining a biologicalsample having subject genomic DNA; extracting the genomic DNA; treatingthe genomic DNA, or a fragment thereof, with one or more reagents toconvert 5-position unmethylated cytosine bases to uracil or to anotherbase that is detectably dissimilar to cytosine in terms of hybridizationproperties; contacting the treated genomic DNA, or the treated fragmentthereof, with an amplification enzyme and at least two primerscomprising, in each case a contiguous sequence at least 9 nucleotides inlength that is complementary to, or hybridizes under moderatelystringent or stringent conditions to a sequence selected from the groupconsisting bisulfite converted sense and antisense strands, andcomplements thereof, wherein the treated DNA or the fragment thereof iseither amplified to produce an amplificate, or is not amplified; anddetermining, based on a presence, absence or class of, or on a propertyof said amplificate, the methylation state or an average, or a valuereflecting an average of the methylation level of at least one, but morepreferably a plurality of CpG dinucleotides of the genomic sequences.

The methods described herein comprise use of at least one methodselected from the group consisting of: i) hybridizing at least onenucleic acid molecule comprising a contiguous sequence at least 9, atleast 25 or at least 50 nucleotides in length that is complementary to,or hybridizes under moderately stringent or stringent conditions to asequence selected from the group consisting of bisulfite converted senseand antisense strands, and complements thereof; ii) hybridizing at leastone nucleic acid molecule, bound to a solid phase, comprising acontiguous sequence at least 9 nucleotides at least 25 or at least 50 inlength that is complementary to, or hybridizes under moderatelystringent or stringent conditions to a sequence selected from the groupconsisting of bisulfite converted sense and antisense strands, andcomplements thereof; iii) hybridizing at least one nucleic acid moleculecomprising a contiguous sequence at least 9, at least 25 or at least 50nucleotides in length that is complementary to, or hybridizes undermoderately stringent or stringent conditions to a sequence selected fromthe group consisting of bisulfite converted sense and antisense strands,and complements thereof, and extending at least one such hybridizednucleic acid molecule by at least one nucleotide base; and iv)sequencing of the amplificate.

Further embodiments provide a method for the analysis (i.e. determiningdisease progression and/or patient prognosis) of a cancer, comprising:obtaining a biological sample having subject genomic DNA; extracting thegenomic DNA; contacting the genomic DNA, or a fragment thereof;comprising one or more sequences selected from the group consisting ofthe genomic sequences or a sequence that hybridizes under stringentconditions thereto, with one or more methylation-sensitive restrictionenzymes, wherein the genomic DNA is either digested thereby to producedigestion fragments, or is not digested thereby; and determining, basedon a presence, absence or class of, or on property of at least one suchfragment, the methylation state of at least one CpG dinucleotidesequence of the genomic sequences or an average, or a value reflectingan average methylation state of a plurality of CpG dinucleotidesequences thereof. The digested or undigested genomic DNA can beamplified prior to said determining. Additional embodiments providenovel genomic and chemically modified nucleic acid sequences, as well asoligonucleotides and/or PNA-oligomers for analysis of cytosinemethylation patterns within the genomic sequences.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-4 show Boxplots of ratios of Septin9 DNA post/pre surgery (pgmethylated Sept9 DNA post surgery divided pg methylated Sept9 DNA presurgery) in colorectal cancer patients sorted by cancer stage (x-axis).Ratios were only plotted for patients showing Septin9 DNA levels>0 presurgery. The four numbers on top of the plot are p-values from one sidedt-tests using levels post versus pre surgery paired by patients. InFIGS. 1 and 3 Stage I: dotted line and circles, Stage II: dashed lineand triangle, Stage III: dashed/dotted line and crosses and Stage IV:closed line and rhombi.

FIGS. 5 and 6 show levels of Septin9 DNA (y-axis: log10 of pg methylatedSept9 DNA) in colorectal cancer patients pre and post surgery (x-axis).The different stages of the cancer were visualized as follows. FIG. 5:Stage I (4 patients): dotted line and circles; stage II (9 patients):dashed line and triangle; Stage III (4 patients): dashed/dotted line andcrosses; Stage IV (2 patients): closed line and rhombus.

FIGS. 7 and 8 show Boxplots of ratios of RASSF2A DNA post/pre surgery(pg methylated Scpt9 DNA post surgery divided pg methylated RASSF2A DNApre surgery) in colorectal cancer patients sorted by cancer stage(x-axis). Ratios were only plotted for patients showing RASSF2A DNAlevels >0 pre surgery. The four numbers on top of the plot are p-valuesfrom one sided paired t-tests using levels post versus pre surgerypaired by patients. FIG. 5:

Stage I (4 patients): dotted line and circles; stage II (9 patients):dashed line and triangle; Stage III (4 patients): dashed/dotted line andcrosses; Stage IV (2 patients): closed line and rhombus.

FIGS. 9-12 show levels of methylated RASSF2A DNA (y-axis: log10 of pgmethylated RASSF2A DNA) in colorectal cancer patients pre and postsurgery (as given on the x-axis). The different stages of the cancerwere visualized as follows. Stage I (4 patients): dotted line andcircles; stage II (9 patients): dashed line and triangle; Stage III (4patients): dashed/dotted line and crosses; Stage IV (2 patients): closedline and rhombus.

FIG. 13 shows the location of the SEPT9 gene within the human genome onchromosome 17q25 (Ensembl Jul 2005). Arrows indicating the location ofSEQ ID NO: 2 and 3.

FIG. 14 shows the quantitative Analysis of Septin9 Methylation in Pre-and Post Surgery Plasma from CRC patients.

FIG. 15 shows quantitative Analysis of RASSF2A Methylation in Pre- andPost Surgery Plasma from CRC Patients.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The term “Observed/Expected Ratio” (“O/E Ratio”) refers to the frequencyof CpG dinucleotides within a particular DNA sequence, and correspondsto the [number of CpG sites/(number of C bases×number of G bases)]/bandlength for each fragment.

The term “CpG island” refers to a contiguous region of genomic DNA thatsatisfies the criteria of (1) having a frequency of CpG dinucleotidescorresponding to an “Observed/Expected Ratio” >0.6, and (2) having a “GCContent” >0.5. CpG islands are typically, but not always, between about0.2 to about 1 KB, or to about 2 kb in length.

The term “methylation state” or “methylation status” refers to thepresence, absence or class of 5-methylcytosine (“5-mCyt”) at one or aplurality of CpG dinucleotides within a DNA sequence. Methylation statesat one or more particular CpG methylation sites (each having two CpGdinucleotide sequences) within a DNA sequence include “unmethylated,”“fully-methylated” and “hemi-methylated.”

The term “hemi-methylation” or “hemimethylation” refers to themethylation state of a double stranded DNA wherein only one strandthereof is methylated.

The term ‘AUC’ as used herein is an abbreviation for the arca under acurve. In particular it refers to the area under a Receiver OperatingCharacteristic (ROC) curve. The ROC curve is a plot of the true positiverate against the false positive rate for the different possible cutpoints of a diagnostic test. It shows the trade-off between sensitivityand specificity depending on the selected cut point (any increase insensitivity will be accompanied by a decrease in specificity). The areaunder an ROC curve (AUC) is a measure for the accuracy of a test (thelarger the area the better, optimum is 1, a random test would have a ROCcurve lying on the diagonal with an area of 0.5; for reference: J. P.Egan. Signal Detection Theory and ROC Analysis, Academic Press, NewYork, 1975).

The term “microarray” refers broadly to both “DNA microarrays,” and ‘DNAchip(s),’ as recognized in the art, encompasses all art-recognized solidsupports, and encompasses all methods for affixing nucleic acidmolecules thereto or synthesis of nucleic acids thereon.

“Genetic parameters” are mutations and polymorphisms of genes andsequences further required for their regulation. To be designated asmutations are, in particular, insertions, deletions, point mutations,inversions and polymorphisms and, particularly preferred, SNPs (singlenucleotide polymorphisms).

“Epigenetic parameters” are, in particular, cytosine methylation.Further epigenetic parameters include, for example, the acetylation ofhistones which, however, cannot be directly analysed using the describedmethod but which, in turn, correlate with the DNA methylation.

The term “bisulfite reagent” refers to a reagent comprising bisulfite,disulfite, hydrogen sulfite or combinations thereof, useful as disclosedherein to distinguish between methylated and unmethylated CpGdinucleotide sequences.

The term “Methylation assay” refers to any assay for determining themethylation state of one or more CpG dinucleotide sequences within asequence of DNA.

The term “MS.AP-PCR” (Methylation-Sensitive Arbitrarily-PrimedPolymerase Chain Reaction) refers to the art-recognized technology thatallows for a global scan of the genome using CG-rich primers to focus onthe regions most likely to contain CpG dinucleotides, and described byGonzalgo et al., Cancer Research 57: 594-599, 1997.

The term “MethyLight™” refers to the art-recognized fluorescence-basedreal-time PCR technique described by Eads et al., Cancer Res. 59:2302-2306, 1999.

The term “HeavyMethyl™” assay, in the embodiment thereof implementedherein, refers to an assay, wherein methylation specific blocking probes(also referred to herein as blockers) covering CpG positions between, orcovered by the amplification primers enable methylation-specificselective amplification of a nucleic acid sample.

The term “HeavyMethyl™ MethyLight™” assay, in the embodiment thereofimplemented herein, refers to a HeavyMethyl™ MethyLight™ assay, which isa variation of the MethyLight™ assay, wherein the MethyLight™ assay iscombined with methylation specific blocking probes covering CpGpositions between the amplification primers.

The term “Ms-SNuPE” (Methylation-sensitive Single Nucleotide PrimerExtension) refers to the art-recognized assay described by Gonzalgo &Jones, Nucleic Acids Res. 25: 2529-2531, 1997.

The term “MSP” (Methylation-specific PCR) refers to the art-recognizedmethylation assay described by Herman et al. Proc. Natl. Acad. Sci. USA93: 9821-9826, 1996, and by U.S. Pat. No. 5,786,146.

The term “COBRA” (Combined Bisulfite Restriction Analysis) refers to theart-recognized methylation assay described by Xiong & Laird, NucleicAcids Res. 25: 2532-2534, 1997.

The term “MCA” (Methylated CpG Island Amplification) refers to themethylation assay described by Toyota et al., Cancer Res. 59: 2307-12,1999, and in WO 00/26401A1.

The term “hybridisation” is to be understood as a bond of anoligonucleotide to a complementary sequence along the lines of theWatson-Crick base pairings in the sample DNA, forming a duplexstructure.

“Stringent hybridisation conditions,” as defined herein, involvehybridising at 68° C. in 5× SSC/5× Denhardt's solution/1.0% SDS, andwashing in 0.2× SSC/0.1% SDS at room temperature, or involve theart-recognized equivalent thereof (e.g., conditions in which ahybridisation is carried out at 60° C. in 2.5× SSC buffer, followed byseveral washing steps at 37° C. in a low buffer concentration, andremains stable). Moderately stringent conditions, as defined herein,involve including washing in 3× SSC at 42° C., or the art-recognizedequivalent thereof The parameters of salt concentration and temperaturecan be varied to achieve the optimal level of identity between the probeand the target nucleic acid. Guidance regarding such conditions isavailable in the art, for example, by Sambrook et al., 1989, MolecularCloning, A Laboratory Manual, Cold Spring Harbor Press, N.Y.; andAusubel et al. (eds.), 1995, Current Protocols in Molecular Biology,(John Wiley & Sons, N.Y.) at Unit 2.10.

The terms “Methylation-specific restriction enzymes” or“methylation-sensitive restriction enzymes” shall be taken to mean anenzyme that selectively digests a nucleic acid dependant on themethylation state of its recognition site. In the case of suchrestriction enzymes which specifically cut if the recognition site isnot methylated or hemimethylated, the cut will not take place, or with asignificantly reduced efficiency, if the recognition site is methylated.In the case of such restriction enzymes which specifically cut if therecognition site is methylated, the cut will not take place, or with asignificantly reduced efficiency if the recognition site is notmethylated. Preferred are methylation-specific restriction enzymes, therecognition sequence of which contains a CG dinucleotide (for instancecgcg or cccggg). Further preferred for some embodiments are restrictionenzymes that do not cut if the cytosine in this dinucleotide ismethylated at the carbon atom C5.

“Non-methylation-specific restriction enzymes” or“non-methylation-sensitive restriction enzymes” are restriction enzymesthat cut a nucleic acid sequence irrespective of the methylation statewith nearly identical efficiency. They are also called“methylation-unspecific restriction enzymes.”

In reference to composite array sequences, the phrase “contiguousnucleotides” refers to a contiguous sequence region of any individualcontiguous sequence of the composite array, but does not include aregion of the composite array sequence that includes a “node,” asdefined herein above.

The description of a biomarker that is methylated in cancer, butunmethylated in non-cancerous tissue as a prognostic indicator of cancershall be taken to include all transcript variants thereof and allpromoter and regulatory elements thereof. Furthermore as a plurality ofSNPs arc known within the biomarker or gene the term shall be taken toinclude all sequence variants thereof.

Overview

The present invention provides a method for determining the prognosis ofa subject having cancer, comprising determining the methylation and/orexpression levels of at least one biomarker that is methylated incancer, but unmethylated in non-cancerous tissue in a biological sampleisolated from said subject wherein methylation and/or expression statusis indicative of the prognosis of said subject having cancer.

Methods for determining the prognosis, and thus the methods and agentsfor treatment of a cancer patient include determining the staging of thetumor based on various criteria. Often this determination includesinvasive procedures to observe histological changes in tissue morphologyand level of invasion of the tumor into neighboring tissue andmetastasis. Various cancer staging or classification methods are used toevaluate the progression or status of the cancer using standardclassification criteria.

In colorectal cancer, two of these staging methods are theTumor-Node-Metastais (TNM) staging (Stages I-IV) as developed by theAmerican Joint Committee on Cancer (AJCC Cancer Staging Manual, 6thEdition, Springer-Verlag, New York, 2002), incorporated herein forreference, and the modified Duke's or Astler-Coller staging system(Stages A-D) (Astler V B, Coller F A., Ann Surg 1954; 139: 846-52). Bothmethods relate measures of the spread of the primary tumor throughlayers of colon or rectal wall to the adjacent organs, lymph nodes anddistant sites to evaluate tumor progression. Estimates of recurrencerisk and treatment decisions in colon cancer are currently basedprimarily on tumor staging.

The invention provides methods and kits for determining the prognosis ofa cancer subject, determining medical treatment for a cancer subject,determining if a tumor from a cancer subject indicates that the tumor isaggressive or has metastatic potential or indicates a reduced survivaltime for the subject, detecting an aggressive form of cancer in asubject, selecting a cancer subject for cancer treatment, or determiningtumor load or cancer burden in a subject comprising determining themethylation and/or expression levels of at least one biomarker that ismethylated in cancer, but unmethylated in non-cancerous tissue in abiological sample isolated from said subject wherein methylation and/orexpression status is indicative of the prognosis of said subject havingcancer. The methods comprise extracting or otherwise isolating thegenomic DNA or fragment thereof from the biological samples; treatingthe extracted or isolated genomic DNA or a fragment thereof with one ormore reagents to convert cytosine bases that are unmethylated in the5-position thereof to uracil or to another base that is detectablydissimilar to cytosine in terms of hybridization properties; contactingthe treated genomic DNA or treated fragment, with an amplificationenzyme and at least one primer comprising, a contiguous sequence of atleast 9, at least 18, at least 25 or at least 50 nucleotides that iscomplementary to, or hybridizes under moderately stringent or stringentconditions to a the treated sequence or to a complement thereof, whereinthe treated genomic DNA or the fragment thereof is either amplified toproduce at least one amplificate, or is not amplified; and determining,based on a presence, absence or amount of, or on a property of saidamplificate, the methylation state or level of at least one CpGdinucleotide of the gene, or an average, or a value reflecting anaverage methylation state or level of a plurality of CpG dinucleotidesof the gene.

Methods of treating the extracted DNA, amplifying the DNA, and detectingthe DNA, and analyzing the DNA are further described herein.

The invention provides the detection in a biological sample isolatedfrom a cancer subject of a biomarker or gene that is methylated incancer, but unmethylated in non-cancerous tissue, and the furtherprognosis, determination of clinical outcome, or determination ofmedical treatment for the cancer subject.

Preferably, the method of the invention comprises the steps of a)measuring the level of methylated genomic DNA of a gene, or a fragmentthereof, in a first biological sample obtained from a subject sufferingfrom cancer; b) measuring the level of methylated genomic DNA of thegene or a fragment thereof, in a further biological sample obtained fromthe subject;

and c) comparing the measured levels of methylated DNA in the furthersample and the first sample.

In an embodiment, the detection and analysis is performed in apre-treatment sample and again in a post-treatment sample, wherein thetreatment is any treatment of the patient (or patient tissue) with aprocedure or administration that would diminish, remove, shrink,minimize or ablate the tumor. Such methods include, but are not limitedto, surgical resection, immunotherapy, radiation therapy, chemotherapy,solid tumor targeting therapies, laser therapy, soft tissue targetingtherapies, and blood cancer treatments. In this embodiment, the“pre-treatment sample” corresponds to the “first sample” and the“post-treatment-sample” corresponds to the “further sample”

The pre-treatment sample may be taken any time before treatmentcommences. However, it is preferably taken not more than 1 week, notmore than 2 weeks, not more than 4 weeks or not more than 8 weeks beforetreatment commences. The post-treatment sample is, preferably taken anytime after the treatment commences. If the treatment is chemotherapy, itis explicitly envisaged that the post-treatment sample is taken beforethe patient's course of treatment is completed provided that the patienthas received at least one dosage of at least one pharmaceutical compoundused for chemotherapy.

The recommended treatment of colon cancer depends on the staging of thetumor. Stages I, II and III are characterized by the absence of distantmetastases. Therefore, surgical resection of the tumor is the treatmentof choice. For stages II B, II C, III and high risk II A adjuvantchemotherapy may be recommended. For stage IV tumors surgical resectionis only recommended if the number and location of distant metastasesindicates the chance of a complete cure by removing all tumors. In stageIV disease surgical resection is accompanied by adjuvant and/orneoadjuvant chemotherapy.

In a preferred embodiment of the present invention the tumor is stage I,II or III colon carcinoma. In this case, a level of the methylatedgenomic DNA in the post-treatment sample which indicates a completeremoval of the tumor, preferably a level below the limit of detection,indicates that the treatment of the cancer by surgical resection wassuccessful. This is equivalent to a good prognosis of the patient andadjuvant chemotherapy as additional treatment is, preferably, notrecommended.

In another preferred embodiment of the present invention, the treatmentis adjuvant or neoadjuvant chemotherapy of a stage I, II or III tumor oran operable stage IV tumor or chemotherapy without additional surgery assystemic treatment of an inoperable stage IV tumor. In this case, adecreased level of the methylated genomic DNA in the post-treatmentsample as compared to the pre-treatment sample, preferably, indicatesthat the selected chemotherapeutic treatment regimen was successful inreducing the tumor burden of the patient. This is equivalent to theindication that the chemotherapeutic treatment regimen does not need tobe adapted. However, if the level of the methylated genomic sequence inthe post-treatment sample remains constant or even increases, thisindicates, preferably, the current treatment is not successful and thetreatment regimen needs to be adapted. In this embodiment the taking ofmore than one post-treatment sample at different points of time duringchemotherapy is preferred in order to constantly determine whether ornot the chemotherapeutic treatment regimen is still successful.

The invention provides for a method of prognosis of a cancer subjectthat encompasses detection of the tumor cells when the primary tumor hasbeen removed, such as by the methods described above. Thus, theinvention provides for determining if the procedures employed to removethe primary tumor were successful and complete. Moreover, the inventionprovides methods to determine if the tumor has spread. Historically,methods to determine if the tumor had spread relied on pathological andhistological methods of determining lymph node involvement andmetastasis, such as the cancer staging methods described above. With thepresent invention, such parameters as tumor load, cancer burden, tumorspread and/or metastasis can be determined by taking a first sample,from which the genomic DNA is evaluated for the presence of a gene orbiomarker that is methylated in cancer, but unmethylated innon-cancerous tissue, and taking a second sample, from which the genomicDNA is evaluated for the presence of a gene or biomarker that ismethylated in cancer, but unmethylated in non-cancerous tissue, anddetermining if the tumor or cancer cells remain in the subject and thusfurther indicate a need for clinical treatment.

In a preferred embodiment, the presence of detectable levels of thebiomarker after removal of the primary tumor indicate that the tumor hasnot been removed completely. More preferably, this situation indicatesthat the tumor has already spread locally into the surrounding tissue orlymphnodes or systemically into organs other than the colon, rectum orappendix.

In certain embodiments, the detection methods are performedquantitatively, quantitatively in part, qualitatively, qualitatively inpart, or quantitatively in part and qualitatively in part.

In certain embodiments the gene or biomarker that is methylated incancer, but unmethylated in non-cancerous tissue is Septin9. In certainembodiments the gene or biomarker that is methylated in cancer, butunmethylated in non-cancerous tissue is RASSF2A.

The human Septin 9 gene (also known as MLL septin-like fusion protein,MLL septin-like fusion protein MSF-A, Slpa, Eseptin, Msf, septin-likeprotein Ovarian/Breast septin (Ov/Br septin) and Septin D1) is locatedon chromosome 17q25 within contig AC068594.15.1.168501 and is a memberof the Septin gene family. FIG. 13 provides the Ensembl annotation ofthe Septin 9 gene, and shows 4 transcript variants, the Septin 9variants and the Q9HC74 variants (which are truncated versions of theSeptin 9 transcripts). SEQ ID NO: 1 provides the sequence of said gene,comprising regions of both the Septin 9 and Q9HC74 transcripts andpromoter regions. SEQ ID NO:2 and SEQ ID NO:3 are sub-regions thereofthat provide the sequence of CpG rich promoter regions of Septin 9 andQ9HC74 transcripts, respectively. SEQ ID NOs: 4 and 5, are sequences forthe chemically (bisulfite)-treated Septin 9 DNA sense strand and theanti-sense strand, respectively, that correspond to the sequence of SEQID NO:1 (i.e., where CpG dinucleotides are methylated), as shown inTable 1. SEQ ID NOs: 10 and 11, are sequences for the chemically(bisulfite)-treated Septin 9 DNA sense strand and the anti-sense strand,respectively, that correspond to the sequence of SEQ ID NO:1 (i.e.,where CpG dinucleotides are unmethylated), as shown in Table 1. SEQ IDNOs: 6, and 7 are sequences for the chemically (bisulfite)-treatedSeptin 9 DNA sense strand and anti-sense strand, respectively, thatcorrespond to SEQ ID NO:2 (i.e., where CpG dinucleotides aremethylated), as shown in Table 1. SEQ ID NOs: 12, and 13 are sequencesfor the chemically (bisulfite)-treated Septin 9 DNA sense strand andanti-sense strand, respectively, that correspond to SEQ ID NO:2 (i.e.,where CpG dinucleotides are unmethylated) as shown in Table 1. SEQ IDNOs: 8 and 9, are sequences for the chemically (bisulfite)-treatedQ9HC74 DNA sense strand and the anti-sense strand, respectively, thatcorrespond to the sequence of SEQ ID NO:3 (i.e., where CpG dinucleotidesare methylated), as shown in Table 1. SEQ ID NOs: 14 and 15, aresequences for the chemically (bisulfite)-treated Septin 9 DNA sensestrand and the anti-sense strand, respectively, that correspond to thesequence of SEQ ID NO:3 (i.e., where CpG dinucleotides areunmethylated), as shown in Table 1. Septin9 and these variants have alsobeen described in published US Patent Application No: US-2009-0075260,issued as U.S. Pat. No: 7,951,563; published US Patent ApplicationNo:2006-0286576, issued as U.S. Pat. No: 7,749,702; and in published USPatent Application No: US-2011-0039719, all of which are incorporatedherein for reference to SEPTIN9 gene description and sequenceinformation. Additional sequences related to the Septin9 gene aredescribed in the examples and description herein.

In certain embodiments the gene or biomarker that is methylated incancer, but unmethylated in non-cancerous tissue is RASSF2A (SEQ IDNO:16). The RASSF2 gene is located at chromosomal location 20p13, andencodes multiple mRNA transcript isoforms. Members of the Ras proteinfamily are associated with cancer, RASSF2 binds to K-Ras, and expressionof RASSF2 is associated with controlled cell growth. Loss of expressionresults in uninhibited cell proliferation, and accordingly RASSF2 is atumour suppressor gene (Vos et. al. J. Biol. Chem., Vol. 278, Issue 30,28045-28051, Jul. 25, 2003). The RASSF2 gene comprises a CpG denseregion in the gene promoter, spanning the first 2 non-coding exons. Thisregion has been characterised as being co-methylated, and furthermore,methylation thereof has been associated with the development of gastricand colon carcinomas. Hesson et al. (Oncogene. 2005 Jun. 2; 24(24):3987-94.) characterised the CpG island as being co-methylated, by meansof COBRA analysis and bisulfite sequencing of colon cancer cell lines.Furthermore, they confirmed by MSP analysis that 21/30 (70%) of analysedcolon cancer cell lines were methylated within the RASSF2A promoterregion. Further research has indicated that RASSF2 methylation may beassociated with gastric cancer (Endoh et. al Br J. Cancer. 2005 Dec. 12;93(12): 1395-9) and nasopharyngeal cancer (Zhang et. al Int J. Cancer.2007 Jan. 1; 120(1): 32-8). SEQ ID NO:16 provides the sequence ofRASSF2A. SEQ ID NOs: 17 and 18, are sequences for the chemically(bisulfite)-treated RASSF2A DNA sense strand and the anti-sense strand,respectively, that correspond to SEQ ID NO:16 (i.e., where CpGdinucleotides are methylated) as shown in Table 1. SEQ ID NOs: 19 and 20are sequences for the chemically (bisulfite)-treated RASSF2A DNA sensestrand and anti-sense strand, respectively, that correspond to thesequence of SEQ ID NO:16 (i.e., where CpG dinucleotides areunmethylated) as shown in Table 1. The genomic whole gene sequence ofwhich is shown in SEQ ID NO:16, which has been described in published USPatent Application No: US-2010-0092953, which is incorporated herein forreference for RASSF2A gene description and sequence information.

Using the methods of the invention the presence, determinedquantitatively, quantitatively in part, qualitatively, qualitatively inpart, or quantitatively in part and qualitatively in part of the gene orbiomarker in the post-treatment sample in a Stage I or II subjectindicates a poor prognosis or need for more aggressive cancer treatment.

Using the methods of the invention an equivalent or higher level of thegene or biomarker in the post-treatment sample in a Stage III subjectindicates the need for continued monitoring using the methods describedherein to see if there is a tendency of the level of the gene orbiomarker to increase.

The methods of the invention provide not only for detecting a change inthe level of the gene or biomarker in the post-treatment sample, butalso for continued monitoring or surveillance of response to treatmentor efficacy of non-treatment of the subject and can be used to determineif the cancer or tumor is in remission or recurrence.

Bisulfite modification of DNA is an art-recognized tool used to assessCpG methylation status. The most frequently used method for analyzingDNA for the presence of 5-methylcytosine is based upon the reaction ofbisulfite with cytosine whereby, upon subsequent alkaline hydrolysis,cytosine is converted to uracil which corresponds to thymine in its basepairing behavior. Significantly, however, 5-methylcytosine remainsunmodified under these conditions. Consequently, the original DNA isconverted in such a manner that methylcytosine, which originally couldnot be distinguished from cytosine by its hybridization behavior, cannow be detected as the only remaining cytosine using standard,art-recognized molecular biological techniques, for example, byamplification and hybridization, or by sequencing. All of thesetechniques are based on differential base pairing properties, which cannow be fully exploited.

An overview of art-recognized methods for detecting 5-methylcytosine isprovided by Rein, T., et al., Nucleic Acids Res., 26: 2255, 1998.

The bisulfite technique, barring few exceptions (e.g., Zeschnigk M, etal., Eur J Hum Genet. 5: 94-98, 1997), is currently only used inresearch. In general, short, specific fragments of a known gene areamplified subsequent to a bisulfite treatment, and either completelysequenced (Olek & Walter, Nat Genet. 1997 17: 275-6, 1997), subjected toone or more primer extension reactions (Gonzalgo & Jones, Nucleic AcidsRes., 25: 2529-31, 1997; WO 95/00669; U.S. Pat. No. 6,251,594) toanalyse individual cytosine positions, or treated by enzymatic digestion(Xiong & Laird, Nucleic Acids Res., 25: 2532-4, 1997). Detection byhybridisation has also been described in the art (Olek et al., WO99/28498). Additionally, use of the bisulfite technique for methylationdetection with respect to individual genes has been described (Grigg &Clark, Bioessays, 16: 431-6, 1994; Zeschnigk M, et al., Hum Mol Genet.,6: 387-95, 1997; Feil R, et al., Nucleic Acids Res., 22: 695-, 1994;Martin V, et al., Gene, 157: 261-4, 1995; WO 9746705 and WO 9515373).

The present invention provides for the use of the bisulfite technique,in combination with one or more methylation assays, for determination ofthe methylation status of CpG dinucleotide sequences within the genomicsequences . Genomic CpG dinucleotides can be methylated or unmethylated(alternatively known as up- and down-methylated respectively). Howeverthe methods of the present invention are suitable for the analysis ofbiological samples of a heterogeneous nature e.g. a low concentration oftumor cells within a background of blood or ejaculate. Accordingly, whenanalyzing the methylation status of a CpG position within such a samplethe person skilled in the art may use a quantitative assay fordetermining the level (e.g. percent, fraction, ratio, proportion ordegree) of methylation at a particular CpG position as opposed to amethylation state. Accordingly the term methylation status ormethylation state should also be taken to mean a value reflecting thedegree of methylation at a CpG position. Unless specifically stated theterms “hypermethylated” or “upmethylated” shall be taken to mean amethylation level above that of a specified cut-off point, wherein saidcut-off may be a value representing the average or median methylationlevel for a given population, or is preferably an optimized cut-offlevel. The “cut-off” is also referred herein as a “threshold”. In thecontext of the present invention the terms “methylated”,“hypermethylated” or “upmethylated” shall be taken to include amethylation level above the cut-off be zero (0) % (or equivalentsthereof) methylation for all CpG positions within and associated with(e.g. in promoter or regulatory regions) at least one gene or genomicsequence that is methylated in cancer, but unmethylated in non-canceroustissue.

According to the present invention, determination of the methylationstatus of CpG dinucleotide sequences within the genomic sequences haveutility in the determination of the prognosis of a subject havingcancer.

Methylation Assay Procedures. Various methylation assay procedures areknown in the art, and can be used in conjunction with the presentinvention. These assays allow for determination of the methylation stateof one or a plurality of CpG dinucleotides (e.g., CpG islands) within aDNA sequence. Such assays involve, among other techniques, DNAsequencing of bisulfite-treated DNA, PCR (for sequence-specificamplification), Southern blot analysis, and use of methylation-sensitiverestriction enzymes.

For example, genomic sequencing has been simplified for analysis of DNAmethylation patterns and 5-methylcytosine distribution by usingbisulfite treatment (Frommer et al., Proc. Natl. Acad. Sci. USA 89:1827-1831, 1992). Additionally, restriction enzyme digestion of PCRproducts amplified from bisulfite-converted DNA is used, e.g., themethod described by Sadri & Hornsby (Nucl. Acids Res. 24: 5058-5059,1996), or COBRA (Combined Bisulfite Restriction Analysis) (Xiong &Laird, Nucleic Acids Res. 25: 2532-2534, 1997).

COBRA. COBRA™ analysis is a quantitative methylation assay useful fordetermining DNA methylation levels at specific gene loci in smallamounts of genomic DNA (Xiong & Laird, Nucleic Acids Res. 25: 2532-2534,1997). Briefly, restriction enzyme digestion is used to revealmethylation-dependent sequence differences in PCR products of sodiumbisulfite-treated DNA. Methylation-dependent sequence differences arefirst introduced into the genomic DNA by standard bisulfite treatmentaccording to the procedure described by Frommer et al. (Proc. Natl.Acad. Sci. USA 89: 1827-1831, 1992). PCR amplification of the bisulfiteconverted DNA is then performed using primers specific for the CpGislands of interest, followed by restriction endonuclease digestion, gelelectrophoresis, and detection using specific, labeled hybridizationprobes. Methylation levels in the original DNA sample are represented bythe relative amounts of digested and undigested PCR product in alinearly quantitative fashion across a wide spectrum of DNA methylationlevels. In addition, this technique can be reliably applied to DNAobtained from microdissected paraffin-embedded tissue samples.

Typical reagents (e.g., as might be found in a typical COBRA™-based kit)for COBRA™ analysis may include, but are not limited to: PCR primers forspecific gene (or bisulfite treated DNA sequence or CpG island);restriction enzyme and appropriate buffer; gene-hybridizationoligonucleotide; control hybridization oligonucleotide; kinase labelingkit for oligonucleotide probe; and labeled nucleotides. Additionally,bisulfite conversion reagents may include: DNA denaturation buffer;sulfonation buffer; DNA recovery reagents or kits (e.g., precipitation,ultrafiltration, affinity column); desulfonation buffer; and DNArecovery components.

Preferably, assays such as “MethyLight™” (a fluorescence-based real-timePCR technique) (Eads et al., Cancer Res. 59: 2302-2306, 1999), Ms-SNuPE™(Methylation-sensitive Single Nucleotide Primer Extension) reactions(Gonzalgo & Jones, Nucleic Acids Res. 25: 2529-2531, 1997),methylation-specific PCR (“MSP”; Herman et al., Proc. Natl. Acad. Sci.USA 93: 9821-9826, 1996; U.S. Pat. No. 5,786,146), and methylated CpGisland amplification (“MCA”; Toyota et al., Cancer Res. 59: 2307-12,1999) are used alone or in combination with other of these methods.

The “HeavyMethyl™” assay, technique is a quantitative method forassessing methylation differences based on methylation specificamplification of bisulfite treated DNA.

Methylation specific blocking probes (also referred to herein asblockers) covering CpG positions between, or covered by theamplification primers enable methylation-specific selectiveamplification of a nucleic acid sample.

The term “HeavyMethyl™ MethyLight™” assay, in the embodiment thereofimplemented herein, refers to a HeavyMethyl™ MethyLight™ assay, which isa variation of the MethyLight™ assay, wherein the MethyLight™ assay iscombined with methylation specific blocking probes covering CpGpositions between the amplification primers. The HeavyMethyl™ assay mayalso be used in combination with methylation specific amplificationprimers.

Typical reagents (e.g., as might be found in a typical MethyLight™-basedkit) for HeavyMethyl™ analysis may include, but are not limited to: PCRprimers for specific genes (or bisulfite treated DNA sequence or CpGisland); blocking oligonucleotides; optimized PCR buffers anddeoxynucleotides; and Taq polymerase.

MSP. MSP (methylation-specific PCR) allows for assessing the methylationstatus of virtually any group of CpG sites within a CpG island,independent of the use of methylation-sensitive restriction enzymes(Herman et al. Proc. Natl. Acad. Sci. USA 93: 9821-9826, 1996; U.S. Pat.No. 5,786,146). Briefly, DNA is modified by sodium bisulfite convertingall unmethylated, but not methylated cytosines to uracil, andsubsequently amplified with primers specific for methylated versusunmethylated DNA. MSP requires only small quantities of DNA, issensitive to 0.1% methylated alleles of a given CpG island locus, andcan be performed on DNA extracted from paraffin-embedded samples.Typical reagents (e.g., as might be found in a typical MSP-based kit)for MSP analysis may include, but are not limited to: methylated andunmethylated PCR primers for specific gene (or bisulfite treated DNAsequence or CpG island), optimized PCR buffers and deoxynucleotides, andspecific probes.

MethyLight™. The MethyLight™ assay is a high-throughput quantitativemethylation assay that utilizes fluorescence-based real-time PCR(TaqMan™) technology that requires no further manipulations after thePCR step (Eads et al., Cancer Res. 59: 2302-2306, 1999). Briefly, theMethyLight™ process begins with a mixed sample of genomic DNA that isconverted, in a sodium bisulfite reaction, to a mixed pool ofmethylation-dependent sequence differences according to standardprocedures (the bisulfite process converts unmethylated cytosineresidues to uracil). Fluorescence-based PCR is then performed in a“biased” (with PCR primers that overlap known CpG dinucleotides)reaction. Sequence discrimination can occur both at the level of theamplification process and at the level of the fluorescence detectionprocess.

The MethyLight™ assay may be used as a quantitative test for methylationpatterns in the genomic DNA sample, wherein sequence discriminationoccurs at the level of probe hybridization. In this quantitativeversion, the PCR reaction provides for a methylation specificamplification in the presence of a fluorescent probe that overlaps aparticular putative methylation site. An unbiased control for the amountof input DNA is provided by a reaction in which neither the primers, northe probe overlie any CpG dinucleotides. Alternatively, a qualitativetest for genomic methylation is achieved by probing of the biased PCRpool with either control oligonucleotides that do not “cover” knownmethylation sites (a fluorescence-based version of the HeavyMethyl™ andMSP techniques), or with oligonucleotides covering potential methylationsites.

The MethyLight™ process can by used with any suitable probes e.g.“TaqMan®”, Lightcycler® etc. . . . For example, double-stranded genomicDNA is treated with sodium bisulfite and subjected to one of two sets ofPCR reactions using TaqMan® probes; e.g., with MSP primers and/orHeavyMethyl blocker oligonucleotides and TaqMan® probe. The TaqMan®probe is dual-labeled with fluorescent “reporter” and “quencher”molecules, and is designed to be specific for a relatively high GCcontent region so that it melts out at about 10° C. higher temperaturein the PCR cycle than the forward or reverse primers. This allows theTaqMan® probe to remain fully hybridized during the PCRannealing/extension step. As the

Taq polymerase enzymatically synthesizes a new strand during PCR, itwill eventually reach the annealed TaqMan® probe. The Taq polymerase 5′to 3′ endonuclease activity will then displace the TaqMan® probe bydigesting it to release the fluorescent reporter molecule forquantitative detection of its now unquenched signal using a real-timefluorescent detection system.

Typical reagents (e.g., as might be found in a typical MethyLight™ basedkit) for MethyLight™ analysis may include, but are not limited to: PCRprimers for specific gene (or bisulfite treated DNA sequence or CpGisland); TaqMan® or Lightcycler® probes; optimized PCR buffers anddeoxynucleotides; and Taq polymerase.

The QM™ (quantitative methylation) assay is an alternative quantitativetest for methylation patterns in genomic DNA samples, wherein sequencediscrimination occurs at the level of probe hybridization. In thisquantitative version, the PCR reaction provides for unbiasedamplification in the presence of a fluorescent probe that overlaps aparticular putative methylation site. An unbiased control for the amountof input DNA is provided by a reaction in which neither the primers, northe probe overlie any CpG dinucleotides. Alternatively, a qualitativetest for genomic methylation is achieved by probing of the biased PCRpool with either control oligonucleotides that do not “cover” knownmethylation sites (a fluorescence-based version of the HeavyMethyl™ andMSP techniques), or with oligonucleotides covering potential methylationsites.

The QM™ process can by used with any suitable probes e.g. “TaqMan®”Lightcycler® etc . . . in the amplification process. For example,double-stranded genomic DNA is treated with sodium bisulfite andsubjected to unbiased primers and the TaqMan® probe. The TaqMan® probeis dual-labeled with fluorescent “reporter” and “quencher” molecules,and is designed to be specific for a relatively high GC content regionso that it melts out at about 10° C. higher temperature in the PCR cyclethan the forward or reverse primers. This allows the TaqMan® probe toremain fully hybridized during the PCR annealing/extension step. As the

Taq polymerase enzymatically synthesizes a new strand during PCR, itwill eventually reach the annealed TaqMan® probe. The Taq polymerase 5′to 3′ endonuclease activity will then displace the TaqMan® probe bydigesting it to release the fluorescent reporter molecule forquantitative detection of its now unquenched signal using a real-timefluorescent detection system.

Typical reagents (e.g., as might be found in a typical QM™-based kit)for QM™ analysis may include, but are not limited to: PCR primers forspecific gene (or bisulfite treated DNA sequence or CpG island); TaqMan®or Lightcycler® probes; optimized PCR buffers and deoxynucleotides; andTaq polymerase.

Ms-SNuPE. The Ms-SNuPE™ technique is a quantitative method for assessingmethylation differences at specific CpG sites based on bisulfitetreatment of DNA, followed by single-nucleotide primer extension(Gonzalgo & Jones, Nucleic Acids Res. 25: 2529-2531, 1997). Briefly,genomic DNA is reacted with sodium bisulfite to convert unmethylatedcytosine to uracil while leaving 5-methylcytosine unchanged.Amplification of the desired target sequence is then performed using PCRprimers specific for bisulfite-converted DNA, and the resulting productis isolated and used as a template for methylation analysis at the CpGsite(s) of interest. Small amounts of DNA can be analyzed (e.g.,microdissected pathology sections), and it avoids utilization ofrestriction enzymes for determining the methylation status at CpG sites.

Typical reagents (e.g., as might be found in a typical Ms-SNuPE™-basedkit) for Ms-SNuPE™ analysis may include, but are not limited to: PCRprimers for specific gene (or bisulfite treated DNA sequence or CpGisland); optimized PCR buffers and deoxynucleotides; gel extraction kit;positive control primers; Ms-SNuPE™ primers for specific gene; reactionbuffer (for the Ms-SNuPE reaction); and labelled nucleotides.Additionally, bisulfite conversion reagents may include: DNAdenaturation buffer; sulfonation buffer; DNA recovery regents or kit(e.g., precipitation, ultrafiltration, affinity column); desulfonationbuffer; and DNA recovery components.

Novel utility for the detection of a biomarker that is methylated incancer, but unmethylated in non-cancerous tissue as a prognosticindicator of cancer/tumor in blood.

In one aspect the method of the invention comprises the following steps:i) determining the methylation and/or expression of at least one gene orgenomic sequence that is methylated in cancer tissue, but un-methylatedin non-cancer tissue; and ii) determining the prognosis of a subjecthaving cancer. In one embodiment, the steps arc carried out in bodilytissue or blood. In an embodiment, the gene is SEPTIN9 (SEQ ID NOs:1-15,and other sequences as described herein), the genomic sequence of whichis unmethylated in non-cancerous tissue, and methylated in canceroustissue. In another embodiment, the gene is RASSF2A (SEQ ID NOs:16-20,and other sequences as described herein), the genomic sequence of whichis unmethylated in non-cancerous tissue, and methylated in canceroustissue.

The method of the invention may be enabled by means of any analysis ofthe expression of an RNA transcribed therefrom or polypeptide or proteintranslated from said RNA, preferably by means of mRNA expressionanalysis or polypeptide expression analysis. However, in the mostpreferred embodiment of the invention the determination of the prognosisof a subject having cancer, is enabled by means of analysis of themethylation status of at least one gene or genomic sequence, and/orpromoter or regulatory elements of the genomic sequence that isunmethylated in non-cancerous tissue, and methylated in canceroustissue. In other embodiments, the present invention also providesprognostic assays and methods, both quantitative and qualitative fordetecting the expression of one or more of the genes in a subject anddetermining therefrom the prognosis of a subject having cancer in saidsubject. In other embodiments, hyper-methylation and/or under-expressionof one or more of the genes is associated with the progression andaggressiveness of cancer.

In a preferred embodiment the presence of methylated Septin9 DNA in asample prior to surgery above 3 pg/ml is indicative of the presence ofcancer. Preferably, after surgery a negative Septin9 methylation signalis indicative of good prognosis (0 pg/ml methylated Septin9). Preferablyafter surgery the presence of methylated Septin9 of above 0 to 3 pg/mlsample indicates a low risk for the recurrence of cancer. Preferablyafter surgery a methylated Septin9 level from 3 to 30 pg/ml plasma isindicative of a medium risk for the recurrence of cancer. Preferably,after surgery the presence of methylated Septin9 of above 30 pg/mlsample indicates a high risk or recurrence. In a preferred embodimentthe presence of methylated RASSF2A DNA in a sample prior to surgeryabove 3 pg/ml is indicative of the presence of cancer. Preferably, aftersurgery a negative RASSF2A methylation signal is indicative of goodprognosis (0 pg/ml methylated RASSF2A). Preferably after surgery thepresence of methylated RASSF2A of above 0 to 3 pg/ml sample indicates alow risk for the recurrence of cancer. Preferably after surgery amethylated RASSF2A level from 3 to 30 pg/ml plasma is indicative of amedium risk for the recurrence of cancer. Preferably, after surgery thepresence of methylated RASSF2A of above 30 pg/ml sample indicates a highrisk or recurrence. Preferred samples are blood, tumor tissue andplasma. Preferably, the cancer is colorectal cancer.

To detect the presence of mRNA encoding a gene or genomic sequence, asample is obtained from the subject. The sample may be any suitablesample comprising cellular matter of the tumor. Suitable sample typesinclude tissue, blood, plasma, or serum and all possible combinationsthereof. It is preferred that said sample types are blood. The samplemay be treated to extract the RNA contained therein. The resultingnucleic acid from the sample is then analysed. Many techniques are knownin the state of the art for determining absolute and relative levels ofgene expression, commonly used techniques suitable for use in thepresent invention include in situ hybridisation (e.g. FISH), Northernanalysis, RNase protection assays (RPA), microarrays and PCR-basedtechniques, such as quantitative PCR and differential display PCR or anyother nucleic acid detection method. Reversetranscription/polymerisation chain reaction technique (RT-PCR) can beused. The method of RT-PCR is well known in the art (for example, seeWatson and Fleming, supra).

The RT-PCR method can be performed as follows. Total cellular RNA isisolated by, for example, the standard guanidium isothiocyanate methodand the total RNA is reverse transcribed. The reverse transcriptionmethod involves synthesis of DNA on a template of RNA using a reversetranscriptase enzyme and a 3′ end oligonucleotide dT primer and/orrandom hexamer primers. The cDNA thus produced is then amplified bymeans of PCR. (Belyaysky et al, Nucl Acid Res 17: 2919-2932, 1989; Krugand Berger, Methods in Enzymology, Academic Press, N.Y., Vol.152, pp.316-325, 1987 which are incorporated by reference). Further preferred isthe “Real-time” variant of RT-PCR, wherein the PCR product is detectedby means of hybridisation probes (e.g. TaqMan, LightCycler, MolecularBeacons & Scorpion) or SYBR green. The detected signal from the probesor SYBR green is then quantitated either by reference to a standardcurve or by comparing the Ct values to that of a calibration standard.Analysis of housekeeping genes is often used to normalize the results.

In Northern blot analysis total or poly(A)+ mRNA is run on a denaturingagarose gel and detected by hybridisation to a labelled probe in thedried gel itself or on a membrane. The resulting signal is proportionalto the amount of target RNA in the RNA population.

Comparing the signals from two or more cell populations or tissuesreveals relative differences in gene expression levels. Absolutequantitation can be performed by comparing the signal to a standardcurve generated using known amounts of an in vitro transcriptcorresponding to the target RNA. Analysis of housekeeping genes, geneswhose expression levels are expected to remain relatively constantregardless of conditions, is often used to normalize the results,eliminating any apparent differences caused by unequal transfer of RNAto the membrane or unequal loading of RNA on the gel.

The first step in Northern analysis is isolating pure, intact RNA fromthe cells or tissue of interest. Because Northern blots distinguish RNAsby size, sample integrity influences the degree to which a signal islocalized in a single band. Partially degraded RNA samples will resultin the signal being smeared or distributed over several bands with anoverall loss in sensitivity and possibly an erroneous interpretation ofthe data. In Northern blot analysis, DNA, RNA and oligonucleotide probescan be used and these probes are preferably labelled (e.g. radioactivelabels, mass labels or fluorescent labels). The size of the target RNA,not the probe, will determine the size of the detected band, so methodssuch as random-primed labelling, which generates probes of variablelengths, are suitable for probe synthesis. The specific activity of theprobe will determine the level of sensitivity, so it is preferred thatprobes with high specific activities, are used.

In an RNase protection assay, the RNA target and an RNA probe of adefined length are hybridised in solution. Following hybridisation, theRNA is digested with RNases specific for single-stranded nucleic acidsto remove any unhybridized, single-stranded target RNA and probe. TheRNases are inactivated, and the RNA is separated e.g. by denaturingpolyacrylamide gel electrophoresis. The amount of intact RNA probe isproportional to the amount of target RNA in the RNA population. RPA canbe used for relative and absolute quantitation of gene expression andalso for mapping RNA structure, such as intron/exon boundaries andtranscription start sites. The RNase protection assay is preferable toNorthern blot analysis as it generally has a lower limit of detection.

The antisense RNA probes used in RPA are generated by in vitrotranscription of a DNA template with a defined endpoint and aretypically in the range of 50-600 nucleotides. The use of RNA probes thatinclude additional sequences not homologous to the target RNA allows theprotected fragment to be distinguished from the full-length probe. RNAprobes are typically used instead of DNA probes due to the ease ofgenerating single-stranded RNA probes and the reproducibility andreliability of RNA:RNA duplex digestion with RNases (Ausubel et al.2003), particularly preferred are probes with high specific activities.

Particularly preferred is the use of microarrays. The microarrayanalysis process can be divided into two main parts. First is theimmobilization of known gene sequences onto glass slides or other solidsupport followed by hybridisation of the fluorescently labelled cDNA(comprising the sequences to be interrogated) to the known genesimmobilized on the glass slide (or other solid phase). Afterhybridisation, arrays are scanned using a fluorescent microarrayscanner. Analysing the relative fluorescent intensity of different genesprovides a measure of the differences in gene expression.

DNA arrays can be generated by immobilizing presynthesizedoligonucleotides onto prepared glass slides or other solid surfaces. Inthis case, representative gene sequences are manufactured and preparedusing standard oligonucleotide synthesis and purification methods. Thesesynthesized gene sequences arc complementary to the RNA transcript(s) ofat least one gene that is methylated in cancer, but unmethylated innon-cancerous tissue and tend to be shorter sequences in the range of25-70 nucleotides. Alternatively, immobilized oligos can be chemicallysynthesized in situ on the surface of the slide. In situ oligonucleotidesynthesis involves the consecutive addition of the appropriatenucleotides to the spots on the microarray; spots not receiving anucleotide are protected during each stage of the process using physicalor virtual masks. Preferably said synthesized nucleic acids are lockednucleic acids.

In expression profiling microarray experiments, the RNA templates usedare representative of the transcription profile of the cells or tissuesunder study. RNA is first isolated from the cell populations or tissuesto be compared. Each RNA sample is then used as a template to generatefluorescently labelled cDNA via a reverse transcription reaction.Fluorescent labelling of the cDNA can be accomplished by either directlabelling or indirect labelling methods. During direct labelling,fluorescently modified nucleotides (e.g., Cy®3- or Cy®5-dCTP) areincorporated directly into the cDNA during the reverse transcription.Alternatively, indirect labelling can be achieved by incorporatingaminoallyl-modified nucleotides during cDNA synthesis and thenconjugating an N-hydroxysuccinimide (NHS)-ester dye to theaminoallyl-modified cDNA after the reverse transcription reaction iscomplete. Alternatively, the probe may be unlabelled, but may bedetectable by specific binding with a ligand which is labelled, eitherdirectly or indirectly. Suitable labels and methods for labellingligands (and probes) are known in the art, and include, for example,radioactive labels which may be incorporated by known methods (e.g.,nick translation or kinasing). Other suitable labels include but arc notlimited to biotin, fluorescent groups, chemiluminescent groups (e.g.,dioxetanes, particularly triggered dioxetanes), enzymes, antibodies, andthe like.

To perform differential gene expression analysis, cDNA generated fromdifferent

RNA samples are labelled with Cy®3. The resulting labelled cDNA ispurified to remove unincorporated nucleotides, free dye and residualRNA. Following purification, the labelled cDNA samples are hybridised tothe microarray. The stringency of hybridisation is determined by anumber of factors during hybridisation and during the washing procedure,including temperature, ionic strength, length of time and concentrationof formamide. These factors are outlined in, for example, Sambrook etal. (Molecular Cloning: A Laboratory Manual, 2nd ed., 1989). Themicroarray is scanned post-hybridisation using a fluorescent microarrayscanner. The fluorescent intensity of each spot indicates the level ofexpression of the analysed gene; bright spots correspond to stronglyexpressed genes, while dim spots indicate weak expression.

Once the images are obtained, the raw data must be analysed. First, thebackground fluorescence must be subtracted from the fluorescence of eachspot. The data is then normalized to a control sequence, such asexogenously added nucleic acids (preferably RNA or DNA), or ahousekeeping gene panel to account for any non-specific hybridisation,array imperfections or variability in the array set-up, cDNA labelling,hybridisation or washing. Data normalization allows the results ofmultiple arrays to be compared.

Another aspect of the invention relates to a kit for use in determiningthe prognosis of a subject having cancer according to the methods of thepresent invention, said kit comprising: a means for measuring the levelof transcription of at least one gene or genomic sequence gene that ismethylated in cancer, but unmethylated in non-cancerous tissue. In apreferred embodiment the means for measuring the level of transcriptioncomprise oligonucleotides or polynucleotides able to hybridise understringent or moderately stringent conditions to the transcriptionproducts of at least one gene or genomic sequence that is methylated incancer, but unmethylated in non-cancerous tissue. In a most preferredembodiment the level of transcription is determined by techniquesselected from the group of Northern Blot analysis, reverse transcriptasePCR, real-time PCR, RNAse protection, and microarray. In anotherembodiment of the invention the kit further comprises means forobtaining and/or storing a biological sample of the subject. Preferredis a kit, which further comprises a container which is most preferablysuitable for containing the means for measuring the level oftranscription and the biological sample of the subject, and mostpreferably further comprises instructions for use and interpretation ofthe kit results.

In a preferred embodiment the kit comprises (a) a plurality ofoligonucleotides or polynucleotides able to hybridise under stringent ormoderately stringent conditions to the transcription products of atleast one gene or genomic sequence that is methylated in cancer, butunmethylated in non-cancerous tissue (b) a container , preferablysuitable for containing the oligonucleotides or polynucleotides and abiological sample of the subject comprising the transcription productswherein the oligonucleotides or polynucleotides can hybridise understringent or moderately stringent conditions to the transcriptionproducts, (c) means to detect the hybridisation of (b); and optionally,(d) instructions for use and interpretation of the kit results.

The kit may also contain other components such as hybridisation buffer(where the oligonucleotides are to be used as a probe) packaged in aseparate container. Alternatively, where the oligonucleotides are to beused to amplify a target region, the kit may contain, packaged inseparate containers, a polymerase and a reaction buffer optimised forprimer extension mediated by the polymerase, such as PCR. Preferablysaid polymerase is a reverse transcriptase. It is further preferred thatsaid kit further contains an Rnase reagent.

The present invention further provides for methods for the detection ofthe presence of the polypeptide encoded by said gene sequences in asample obtained from said subject.

Aberrant levels of polypeptide expression of the polypeptides encoded atleast one gene or genomic sequence that is methylated in cancer, butunmethylated in non-cancerous tissue are associated with the prognosisof a subject having cancer.

According to the present invention under-expression of said polypeptidesis associated with a negative prognosis of a subject having cancer.

Any method known in the art for detecting polypeptides can be used. Suchmethods include, but are not limited to masss-spectrometry,immunodiffusion, immunoelectrophoresis, immunochemical methods,binder-ligand assays, immunohistochemical techniques, agglutination andcomplement assays (e.g., see Basic and Clinical Immunology, Sites andTerr, eds., Appleton & Lange, Norwalk, Conn. pp 217-262, 1991 which isincorporated by reference). Preferred are binder-ligand immunoassaymethods including reacting antibodies with an epitope or epitopes andcompetitively displacing a labelled polypeptide or derivative thereof.

Certain embodiments of the present invention comprise the use ofantibodies specific to the polypeptide(s) encoded by at least one geneor genomic sequence that is methylated in cancer, but unmethylated innon-cancerous tissue .

Such antibodies are useful for determining the prognosis of a subjecthaving cancer. In certain embodiments production of monoclonal orpolyclonal antibodies can be induced by the use of an epitope encoded bya polypeptide of at least one gene or genomic sequence that ismethylated in cancer, but unmethylated in non-cancerous tissue as anantigene. Such antibodies may in turn be used to detect expressedpolypeptides. The levels of such polypeptides present may be quantifiedby conventional methods. Antibody-polypeptide binding may be detectedand quantified by a variety of means known in the art, such as labellingwith fluorescent or radioactive ligands. The invention further compriseskits for performing the above-mentioned procedures, wherein such kitscontain antibodies specific for the investigated polypeptides.

Numerous competitive and non-competitive polypeptide bindingimmunoassays are well known in the art. Antibodies employed in suchassays may be unlabelled, for example as used in agglutination tests, orlabelled for use a wide variety of assay methods. Labels that can beused include radionuclides, enzymes, fluorescers, chemiluminescers,enzyme substrates or co-factors, enzyme inhibitors, particles, dyes andthe like. Preferred assays include but are not limited toradioimmunoassay (RIA), enzyme immunoassays, e.g., enzyme-linkedimmunosorbent assay (ELISA), fluorescent immunoassays and the like.Polyclonal or monoclonal antibodies or epitopes thereof can be made foruse in immunoassays by any of a number of methods known in the art.

In an alternative embodiment of the method the proteins may be detectedby means of western blot analysis. Said analysis is standard in the art,briefly proteins are separated by means of electrophoresis e.g.SDS-PAGE. The separated proteins arc then transferred to a suitablemembrane (or paper) e.g. nitrocellulose, retaining the spacialseparation achieved by electrophoresis. The membrane is then incubatedwith a blocking agent to bind remaining sticky places on the membrane,commonly used agents include generic protein (e.g. milk protein). Anantibody specific to the protein of interest is then added, saidantibody being detectably labelled for example by dyes or enzymaticmeans (e.g. alkaline phosphatase or horseradish peroxidase). Thelocation of the antibody on the membrane is then detected.

In an alternative embodiment of the method the proteins may be detectedby means of immunohistochemistry (the use of antibodies to probespecific antigens in a sample). Said analysis is standard in the art,wherein detection of antigens in tissues is known asimmunohistochemistry, while detection in cultured cells is generallytermed immunocytochemistry. Briefly the primary antibody to be detectedby binding to its specific antigen. The antibody-antigen complex is thenbound by a secondary enzyme conjugated antibody. In the presence of thenecessary substrate and chromogen the bound enzyme is detected accordingto coloured deposits at the antibody-antigen binding sites. There is awide range of suitable sample types, antigen-antibody affinity, antibodytypes, and detection enhancement methods. Thus optimal conditions forimmunohistochemical or immunocytochemical detection must be determinedby the person skilled in the art for each individual case.

One approach for preparing antibodies to a polypeptide is the selectionand preparation of an amino acid sequence of all or part of thepolypeptide, chemically synthesising the amino acid sequence andinjecting it into an appropriate animal, usually a rabbit or a mouse(Milstein and Kohler Nature 256: 495-497, 1975; Gulfre and Milstein,Methods in Enzymology: Immunochemical Techniques 73: 1-46, Langone andBanatis eds., Academic Press, 1981 which are incorporated by referencein its entirety). Methods for preparation of the polypeptides orepitopes thereof include, but are not limited to chemical synthesis,recombinant DNA techniques or isolation from biological samples.

In the final step of the method the prognosis of the subject isdetermined, whereby under-expression (of mRNA or polypeptides) isindicative of the prognosis of a subject having cancer. The termunder-expression shall be taken to mean expression at a detected levelless than a pre-determined cut off which may be selected from the groupconsisting of the mean, median or an optimised threshold value. The termover-expression shall be taken to mean expression at a detected levelgreater than a pre-determined cut off which may be selected from thegroup consisting of the mean, median or an optimised threshold value.

Another aspect of the invention provides a kit for use in determiningthe prognosis of a subject having cancer according to the methods of thepresent invention, comprising: a means for detecting at least one geneor genomic sequence that is methylated in cancer, but unmethylated innon-cancerous tissue polypeptides. The means for detecting thepolypeptides comprise preferably antibodies, antibody derivatives, orantibody fragments. The polypeptides are most preferably detected bymeans of Western Blotting utilizing a labelled antibody. In anotherembodiment of the invention the kit further comprising means forobtaining a biological sample of the subject. Preferred is a kit, whichfurther comprises a container suitable for containing the means fordetecting the polypeptides in the biological sample of the subject, andmost preferably further comprises instructions for use andinterpretation of the kit results. In a preferred embodiment the kitcomprises: (a) a means for detecting at least one gene or genomicsequence that is methylated in cancer, but unmethylated in non-canceroustissue polypeptides; (b) a container suitable for containing the saidmeans and the biological sample of the subject comprising thepolypeptides wherein the means can form complexes with the polypeptides;(c) a means to detect the complexes of (b); and optionally (d)instructions for use and interpretation of the kit results.

The kit may also contain other components such as buffers or solutionssuitable for blocking, washing or coating, packaged in a separatecontainer.

Methylation Analysis

Particular embodiments of the present invention provide a novelapplication of the analysis of methylation levels and/or patterns withinat least one gene or genomic sequence that is methylated in cancer, butunmethylated in non-cancerous tissue that enables determination of theprognosis of a subject having cancer.

In one embodiment of the method, the prognosis of a subject havingcancer is determined by analysis of the methylation status of one ormore CpG dinucleotides of at least one gene or genomic sequence that ismethylated in cancer, but unmethylated in non-cancerous tissue.

In one embodiment the invention of said method comprises the followingsteps: i) contacting genomic DNA (preferably isolated from tissue,blood, plasma, or serum) obtained from the subject with at least onereagent, or series of reagents that distinguishes between methylated andnon-methylated CpG dinucleotides within at least one gene or genomicsequence that is methylated in cancer, but unmethylated in non-canceroustissue (including promoter and regulatory regions thereof) and ii)determining the prognosis of said subject having cancer.

It is preferred that said one or more CpG dinucleotides of at least onegene or genomic sequence that is methylated in cancer, but unmethylatedin non-cancerous tissue are comprised within a respective genomic targetsequence thereof as provided in the genomic sequences and complementsthereof. The present invention further provides a method forascertaining genetic and/or epigenetic parameters of at least one geneor genomic sequence that is methylated in cancer, but unmethylated innon-cancerous tissue and/or the genomic sequence according to thegenomic sequences within a subject by analysing cytosine methylation.Said method comprising contacting a nucleic acid comprising the genomicsequences in a biological sample obtained from said subject with atleast one reagent or a series of reagents, wherein said reagent orseries of reagents, distinguishes between methylated and non-methylatedCpG dinucleotides within the target nucleic acid.

In a preferred embodiment, said method comprises the following steps: Inthe first step, a sample of the tissue to be analysed is obtained. Thesource may be any suitable source, such as tissue, blood, plasma, orserum and all possible combinations thereof. It is preferred that saidsources of DNA are tissue, blood, plasma, or serum.

The genomic DNA is then isolated from the sample. Genomic DNA may beisolated by any means standard in the art, including the use ofcommercially available kits. Briefly, wherein the DNA of interest isencapsulated in by a cellular membrane the biological sample must bedisrupted and lysed by enzymatic, chemical or mechanical means. The DNAsolution may then be cleared of proteins and other contaminants e.g. bydigestion with proteinase K. The genomic DNA is then recovered from thesolution. This may be carried out by means of a variety of methodsincluding salting out, organic extraction or binding of the DNA to asolid phase support. The choice of method will be affected by severalfactors including time, expense and required quantity of DNA.

Wherein the sample DNA is not enclosed in a membrane (e.g. circulatingDNA from a blood sample) methods standard in the art for the isolationand/or purification of DNA may be employed. Such methods include the useof a protein degenerating reagent e.g. chaotropic salt e.g. guanidinehydrochloride or urea; or a detergent e.g. sodium dodecyl sulphate(SDS), cyanogen bromide. Alternative methods include but are not limitedto ethanol precipitation or propanol precipitation, vacuum concentrationamongst others by means of a centrifuge. The person skilled in the artmay also make use of devices such as filter devices e.g.ultrafiltration, silica surfaces or membranes, magnetic particles,polystyrol particles, polystyrol surfaces, positively charged surfaces,and positively charged membranes, charged membranes, charged surfaces,charged switch membranes, charged switched surfaces.

Once the nucleic acids have been extracted, the genomic double strandedDNA is used in the analysis, methylation analysis may be carried out byany means known in the art including but not limited to methylationsensitive restriction enzyme analysis and chemical reagent analysis.

Chemical Analysis

In the second step of the method, the genomic DNA sample is treated insuch a manner that cytosine bases which are unmethylated at the5′-position are converted to uracil, thymine, or another base which isdissimilar to cytosine in terms of hybridisation behaviour. This will beunderstood as ‘pre-treatment’ or ‘treatment’ herein.

This is preferably achieved by means of treatment with a bisulfitereagent. The term “bisulfite reagent” refers to a reagent comprisingbisulfite, disulfite, hydrogen sulfite or combinations thereof, usefulas disclosed herein to distinguish between methylated and unmethylatedCpG dinucleotide sequences. Methods of said treatment are known in theart (e.g. PCT/EP2004/011715, which is incorporated by reference in itsentirety). It is preferred that the bisulfite treatment is conducted inthe presence of denaturing solvents such as but not limited ton-alkylenglycol, particularly diethylene glycol dimethyl ether (DME), orin the presence of dioxane or dioxane derivatives. In a preferredembodiment the denaturing solvents are used in concentrations between 1%and 35% (v/v). It is also preferred that the bisulfite reaction iscarried out in the presence of scavengers such as but not limited tochromane derivatives, e.g., 6-hydroxy-2,5,7,8,-tetramethylchromane2-carboxylic acid or trihydroxybenzoe acid and derivates thereof, e.g.Gallic acid (see: PCT/EP2004/011715 which is incorporated by referencein its entirety). The bisulfite conversion is preferably carried out ata reaction temperature between 30° C. and 70° C., whereby thetemperature is increased to over 85° C. for short periods of timesduring the reaction (see: PCT/EP2004/011715 which is incorporated byreference in its entirety). The bisulfite treated DNA is preferablypurified priori to the quantification. This may be conducted by anymeans known in the art, such as but not limited to ultrafiltration,preferably carried out by means of Microcon{circumflex over ( )}™columns (manufactured by Millipore{circumflex over ( )}™). Thepurification is carried out according to a modified manufacturer'sprotocol (see: PCT/EP2004/011715 which is incorporated by reference inits entirety).

In the third step of the method, fragments of the treated DNA areamplified, using sets of primer oligonucleotides according to thepresent invention, and an amplification enzyme. The amplification ofseveral DNA segments can be carried out simultaneously in one and thesame reaction vessel. Typically, the amplification is carried out usinga polymerase chain reaction (PCR). Preferably said amplificates are 100to 2,000 base pairs in length. The set of primer oligonucleotidesincludes at least two oligonucleotides whose sequences are each reversecomplementary, identical, or hybridise under stringent or highlystringent conditions to an at least 16-base-pair long segment of thebase sequences of one of the bisulfite sequences and sequencescomplementary thereto.

In an alternate embodiment of the method, the methylation status ofpre-selected CpG positions within at least one gene or genomic sequencethat is methylated in cancer, but unmethylated in non-cancerous tissueand preferably within the nucleic acid sequences according to thegenomic sequences, may be detected by use of methylation-specific primeroligonucleotides. This technique (MSP) has been described in U.S. Pat.No. 6,265,171 to Herman. The use of methylation status specific primersfor the amplification of bisulfite treated DNA allows thedifferentiation between methylated and unmethylated nucleic acids. MSPprimers pairs contain at least one primer which hybridises to abisulfite treated CpG dinucleotide. Therefore, the sequence of saidprimers comprises at least one CpG dinucleotide. MSP primers specificfor non-methylated DNA contain a “T’ at the position of the C positionin the CpG. Preferably, therefore, the base sequence of said primers isrequired to comprise a sequence having a length of at least 9nucleotides which hybridises to a treated nucleic acid sequenceaccording to one of the bisulfite sequences and sequences complementarythereto, wherein the base sequence of said oligomers comprises at leastone CpG dinucleotide. A further preferred embodiment of the methodcomprises the use of blocker oligonucleotides (the HeavyMethyl™ assay).The use of such blocker oligonucleotides has been described by Yu etal., BioTechniques 23: 714-720, 1997. Blocking probe oligonucleotidesare hybridised to the bisulfite treated nucleic acid concurrently withthe PCR primers. PCR amplification of the nucleic acid is terminated atthe 5′ position of the blocking probe, such that amplification of anucleic acid is suppressed where the complementary sequence to theblocking probe is present. The probes may be designed to hybridize tothe bisulfite treated nucleic acid in a methylation status specificmanner. For example, for detection of methylated nucleic acids within apopulation of unmethylated nucleic acids, suppression of theamplification of nucleic acids which are unmethylated at the position inquestion would be carried out by the use of blocking probes comprising a‘CpA’ or ‘TpA’ at the position in question, as opposed to a ‘CpG’ if thesuppression of amplification of methylated nucleic acids is desired.

For PCR methods using blocker oligonucleotides, efficient disruption ofpolymerase-mediated amplification requires that blocker oligonucleotidesnot be elongated by the polymerase. Preferably, this is achieved throughthe use of blockers that are 3′-deoxyoligonucleotides, oroligonucleotides derivitized at the 3′ position with other than a “free”hydroxyl group. For example, 3′-O-acetyl oligonucleotides arerepresentative of a preferred class of blocker molecule.

Additionally, polymerase-mediated decomposition of the blockeroligonucleotides should be precluded. Preferably, such preclusioncomprises either use of a polymerase lacking 5′-3′ exonuclease activity,or use of modified blocker oligonucleotides having, for example, thioatebridges at the 5′-terminii thereof that render the blocker moleculenuclease-resistant. Particular applications may not require such 5′modifications of the blocker. For example, if the blocker- andprimer-binding sites overlap, thereby precluding binding of the primer(e.g., with excess blocker), degradation of the blocker oligonucleotidewill be substantially precluded. This is because the polymerase will notextend the primer toward, and through (in the 5′-3′ direction) theblocker—a process that normally results in degradation of the hybridizedblocker oligonucleotide.

A particularly preferred blocker/PCR embodiment, for purposes of thepresent invention and as implemented herein, comprises the use ofpeptide nucleic acid (PNA) oligomers as blocking oligonucleotides. SuchPNA blocker oligomers are ideally suited, because they are neitherdecomposed nor extended by the polymerase.

Preferably, therefore, the base sequence of said blockingoligonucleotides is required to comprise a sequence having a length ofat least 9 nucleotides which hybridises to a treated nucleic acidsequence according to one of the bisulfite sequences and sequencescomplementary thereto, wherein the base sequence of saidoligonucleotides comprises at least one CpG, TpG or CpA dinucleotide. Itis particularly preferred that the base sequence of said blockingoligonucleotides is required to comprise a sequence having a length ofat least 9 nucleotides which hybridises to a treated nucleic acidsequence according to one of SEQ ID NOs: 5, 6, 9 or 10 and sequencescomplementary thereto, wherein the base sequence of saidoligonucleotides comprises at least one TpG or CpA dinucleotide.

The fragments obtained by means of the amplification can carry adirectly or indirectly detectable label. Preferred are labels in theform of fluorescence labels, radionuclides, or detachable moleculefragments having a typical mass which can be detected in a massspectrometer. Where said labels are mass labels, it is preferred thatthe labelled amplificates have a single positive or negative net charge,allowing for better delectability in the mass spectrometer. Thedetection may be carried out and visualized by means of, e.g., matrixassisted laser desorption/ionization mass spectrometry (MALDI) or usingelectron spray mass spectrometry (ESI).

Matrix Assisted Laser Desorption/Ionization Mass Spectrometry(MALDI-TOF) is a very efficient development for the analysis ofbiomolecules (Karas & Hillenkamp, Anal Chem., 60: 2299-301, 1988). Ananalyte is embedded in a light-absorbing matrix. The matrix isevaporated by a short laser pulse thus transporting the analyte moleculeinto the vapor phase in an unfragmented manner. The analyte is ionizedby collisions with matrix molecules. An applied voltage accelerates theions into a field-free flight tube. Due to their different masses, theions are accelerated at different rates. Smaller ions reach the detectorsooner than bigger ones. MALDI-TOF spectrometry is well suited to theanalysis of peptides and proteins. The analysis of nucleic acids issomewhat more difficult (Gut & Beck, Current Innovations and FutureTrends, 1: 147-57, 1995). The sensitivity with respect to nucleic acidanalysis is approximately 100-times less than for peptides, anddecreases disproportionally with increasing fragment size. Moreover, fornucleic acids having a multiply negatively charged backbone, theionization process via the matrix is considerably less efficient. InMALDI-TOF spectrometry, the selection of the matrix plays an eminentlyimportant role. For desorption of peptides, several very efficientmatrixes have been found which produce a very fine crystallisation.There are now several responsive matrixes for DNA, however, thedifference in sensitivity between peptides and nucleic acids has notbeen reduced. This difference in sensitivity can be reduced, however, bychemically modifying the DNA in such a manner that it becomes moresimilar to a peptide. For example, phosphorothioate nucleic acids, inwhich the usual phosphates of the backbone are substituted withthiophosphates, can be converted into a charge-neutral DNA using simplealkylation chemistry (Gut & Beck, Nucleic Acids Res. 23: 1367-73, 1995).The coupling of a charge tag to this modified DNA results in an increasein MALDI-TOF sensitivity to the same level as that found for peptides. Afurther advantage of charge tagging is the increased stability of theanalysis against impurities, which makes the detection of unmodifiedsubstrates considerably more difficult.

In the fourth step of the method, the amplificates obtained during thethird step of the method are analysed in order to ascertain themethylation status of the CpG dinucleotides prior to the treatment.

In embodiments where the amplificates were obtained by means of MSPamplification, the presence, absence or class of an amplificate is initself indicative of the methylation state of the CpG positions coveredby the primer, according to the base sequences of said primer.

Amplificates obtained by means of both standard and methylation specificPCR may be further analysed by means of based-based methods such as, butnot limited to, array technology and probe based technologies as well asby means of techniques such as sequencing and template directedextension.

In one embodiment of the method, the amplificates synthesised in stepthree are subsequently hybridized to an array or a set ofoligonucleotides and/or PNA probes. In this context, the hybridizationtakes place in the following manner: the set of probes used during thehybridization is preferably composed of at least 2 oligonucleotides orPNA-oligomers; in the process, the amplificates serve as probes whichhybridize to oligonucleotides previously bonded to a solid phase; thenon-hybridized fragments are subsequently removed; said oligonucleotidescontain at least one base sequence having a length of at least 9nucleotides which is reverse complementary or identical to a segment ofthe base sequences specified in the present Sequence Listing; and thesegment comprises at least one CpG , TpG or CpA dinucleotide. Thehybridizing portion of the hybridizing nucleic acids is typically atleast 9, 15, 20, 25, 30 or 35 nucleotides in length. However, longermolecules have inventive utility, and are thus within the scope of thepresent invention.

In a preferred embodiment, said dinucleotide is present in the centralthird of the oligomer. For example, wherein the oligomer comprises oneCpG dinucleotide, said dinucleotide is preferably the fifth to ninthnucleotide from the 5′-end of a 13-mer. One oligonucleotide exists forthe analysis of each CpG dinucleotide within a sequence selected fromthe group consisting the genomic sequences , and the equivalentpositions within the bisulfite sequences. Said oligonucleotides may alsobe present in the form of peptide nucleic acids. The non-hybridisedamplificates are then removed. The hybridised amplificates are thendetected. In this context, it is preferred that labels attached to theamplificates are identifiable at each position of the solid phase atwhich an oligonucleotide sequence is located.

In yet a further embodiment of the method, the genomic methylationstatus of the CpG positions may be ascertained by means ofoligonucleotide probes (as detailed above) that are hybridised to thebisulfite treated DNA concurrently with the PCR amplification primers(wherein said primers may either be methylation specific or standard).

A particularly preferred embodiment of this method is the use offluorescence-based Real Time Quantitative PCR (Heid et al., Genome Res.6: 986-994, 1996; also see U.S. Pat. No. 6,331,393) employing adual-labelled fluorescent oligonucleotide probe (TaqMan™ PCR, using anABI Prism 7700 Sequence Detection System, Perkin Elmer AppliedBiosystems, Foster City, Calif.). The TaqMan™ PCR reaction employs theuse of a non-extendible interrogating oligonucleotide, called a TaqMan™probe, which, in preferred embodiments, is designed to hybridise to aCpG-rich sequence located between the forward and reverse amplificationprimers. The TaqMan™ probe further comprises a fluorescent “reportermoiety” and a “quencher moiety” covalently bound to linker moieties(e.g., phosphoramidites) attached to the nucleotides of the TaqMan™oligonucleotide. For analysis of methylation within nucleic acidssubsequent to bisulfite treatment, it is required that the probe bemethylation specific, as described in U.S. Pat. No. 6,331,393, (herebyincorporated by reference in its entirety) also known as the MethyLight™assay. Variations on the TaqMan™ detection methodology that are alsosuitable for use with the described invention include the use ofdual-probe technology (LightCycler™) or fluorescent amplificationprimers (Sunrise™ technology). Both these techniques may be adapted in amanner suitable for use with bisulfite treated DNA, and moreover formethylation analysis within CpG dinucleotides.

In a further preferred embodiment of the method, the fourth step of themethod comprises the use of template-directed oligonucleotide extension,such as MS-SNuPE as described by Gonzalgo & Jones, Nucleic Acids Res.25: 2529-2531, 1997.

In yet a further embodiment of the method, the fourth step of the methodcomprises sequencing and subsequent sequence analysis of the amplificategenerated in the third step of the method (Sanger F., et al., Proc NatlAcad Sci USA 74: 5463-5467, 1977).

In the an embodiment of the method the genomic nucleic acids areisolated and treated according to the first three steps of the methodoutlined above, namely:

a) obtaining, from a subject, a biological sample having subject genomicDNA;b) extracting or otherwise isolating the genomic DNA;c) treating the genomic DNA of b), or a fragment thereof, with one ormore reagents to convert cytosine bases that are unmethylated in the5-position thereof to uracil or to another base that is detectablydissimilar to cytosine in terms of hybridization properties; and wherein) amplifying subsequent to treatment in c) is carried out in amethylation specific manner, namely by use of methylation specificprimers or blocking oligonucleotides, and further whereine) detecting of the amplificates is carried out by means of a real-timedetection probe, as described above, andf) determining a prognosis for the subject.g) Preferably, where the subsequent amplification of d) is carried outby means of methylation specific primers, as described above, saidmethylation specific primers comprise a sequence having a length of atleast 9, at least 6, at least 25, or at least 50 nucleotides whichhybridises to a treated nucleic acid sequence according to one of thebisulfite sequences and sequences complementary thereto, wherein thebase sequence of said oligomers comprise at least one CpG dinucleotide.

Step e) of the method, namely the detection of the specific amplificatesindicative of the methylation status of one or more CpG positionsaccording to the genomic sequences is carried out by means of real-timedetection methods as described above.

Methylation Sensitive Restriction Enzyme Analysis

In an alternative embodiment of the invention the above described secondstep may be carried out by means of methylation sensitive or methylationspecific restriction enzyme analysis. Methods are known in the artwherein a methylation sensitive restriction enzyme reagent, or a seriesof restriction enzyme reagents comprising methylation sensitiverestriction enzyme reagents that distinguishes between methylated andnon-methylated CpG dinucleotides within a target region are utilized indetermining methylation, for example but not limited to DMH.

In a preferred embodiment, the DNA may be cleaved prior to treatmentwith methylation sensitive restriction enzymes. Such methods are knownin the art and may include both physical and enzymatic means.Particularly preferred is the use of one or a plurality of restrictionenzymes which are not methylation sensitive, and whose recognition sitesare AT rich and do not comprise CG dinucleotides. The use of suchenzymes enables the conservation of CpG islands and CpG rich regions inthe fragmented DNA. The non-methylation-specific restriction enzymes arepreferably selected from the group consisting of Msel, BfaI, Csp6I,Tru1I, Tvu1I, Tru9I, Tvu9I, MaeI and XspI. Particularly preferred is theuse of two or three such enzymes. Particularly preferred is the use of acombination of MseI, BfaI and Csp6I.

The fragmented DNA may then be ligated to adaptor oligonucleotides inorder to facilitate subsequent enzymatic amplification. The ligation ofoligonucleotides to blunt and sticky ended DNA fragments is known in theart, and is carried out by means of dephosphorylation of the ends (e.g.using calf or shrimp alkaline phosphatase) and subsequent ligation usingligase enzymes (e.g. T4 DNA ligase) in the presence of dATPs. Theadaptor oligonucleotides are typically at least 18 base pairs in length.

In the third step, the DNA (or fragments thereof) is then digested withone or more methylation sensitive restriction enzymes. The digestion iscarried out such that hydrolysis of the DNA at the restriction site isinformative of the methylation status of a specific CpG dinucleotide ofat least one gene or genomic sequence that is methylated in cancer, butunmethylated in non-cancerous tissue .

Preferably, the methylation-specific restriction enzyme is selected fromthe group consisting of Bsi E1, Hga I HinP1, Hpy99I, Ava I, Bce AI, BsaHI, BisI, BstUI, BshI236I, AccII, BstFNI, McrBC, GlaI, MvnI, HpaII(HapII), HhaI, AciI, SmaI, HinP1I, HpyCH4IV, EagI and mixtures of two ormore of the above enzymes. Preferred is a mixture containing therestriction enzymes BstUI, HpaII, HpyCH4IV and HinP1I.

In the fourth step, which is optional but a preferred embodiment, therestriction fragments are amplified. This is preferably carried outusing a polymerase chain reaction, and said amplificates may carrysuitable detectable labels as discussed above, namely fluorophorelabels, radionuclides and mass labels. Particularly preferred isamplification by means of an amplification enzyme and at least twoprimers comprising, in each case a contiguous sequence at least 16nucleotides in length that is complementary to, or hybridizes undermoderately stringent or stringent conditions to a sequence selected fromthe group consisting the genomic sequences , and complements thereof.Preferably said contiguous sequence is at least 16, 20 or 25 nucleotidesin length. In an alternative embodiment said primers may becomplementary to any adaptors linked to the fragments.

In the fifth step the amplificates are detected. The detection may be byany means standard in the art, for example, but not limited to, gelelectrophoresis analysis, hybridisation analysis, incorporation ofdetectable tags within the PCR products, DNA array analysis, MALDI orESI analysis. Preferably said detection is carried out by hybridisationto at least one nucleic acid or peptide nucleic acid comprising in eachcase a contiguous sequence at least 16 nucleotides in length that iscomplementary to, or hybridizes under moderately stringent or stringentconditions to a sequence selected from the group consisting the genomicsequences , and complements thereof. Preferably said contiguous sequenceis at least 16, 20 or 25 nucleotides in length.

Subsequent to the determination of the methylation state or level of thegenomic nucleic acids the prognosis of a subject having cancer, isdeduced based upon the methylation state or level of at least one CpGdinucleotide sequence that is methylated in cancer, but unmethylated innon-cancerous tissue, or an average, or a value reflecting an averagemethylation state of a plurality of CpG dinucleotide sequences of thegenomic sequences wherein methylation is associated with the prognosisof a subject having cancer. Wherein said methylation is determined byquantitative means the cut-off point for determining said presence ofmethylation is preferably zero (i.e. wherein a sample displays anydegree of methylation it is determined as having a methylated status atthe analysed CpG position). Nonetheless, it is foreseen that the personskilled in the art may wish to adjust said cut-off value in order toprovide an assay of a particularly preferred sensitivity or specificity.Accordingly said cut-off value may be increased (thus increasing thespecificity), said cut off value may be within a range selected form thegroup consisting of 0%-5%, 5%-10%, 10%-15%, 15%-20%, 20%-30% and30%-50%. Particularly preferred are cut-offs that are at least 0.1%, 1%,10%, 15%, 25%, and 30%.

As used herein the term “prognosis” shall be taken to mean an indicatorof the predicted progression of the disease (including but not limitedto aggressiveness and metastatic potential) and/or predicted patientsurvival time.

In the context of the present invention the term ‘aggressiveness’ istaken to mean one or more of high likelihood of relapse post surgery;below average or below median patient survival; below average or belowmedian disease free survival; below average or below median relapse-freesurvival; above average tumor-related complications; fast progression oftumor or metastases.

Unless stated otherwise as used herein the term “survival” shall betaken to include all of the following: survival until mortality, alsoknown as overall survival (wherein said mortality may be eitherirrespective of cause or tumor related); “recurrence-free survival”(wherein the term recurrence shall include both localized and distantrecurrence) ; metastasis free survival;

disease free survival (wherein the term disease shall include cancer anddiseases associated therewith). The length of said survival may becalculated by reference to a defined start point (e.g. time of diagnosisor start of treatment) and end point (e.g. death, recurrence ormetastasis).

The disclosed invention provides treated nucleic acids, derived from thegenomic sequences, wherein the treatment is suitable to convert at leastone unmethylated cytosine base of the genomic DNA sequence to uracil oranother base that is detectably dissimilar to cytosine in terms ofhybridization for use in determining prognosis of a subject havingcancer or a tumor. The genomic sequences in question may comprise one,or more consecutive methylated CpG positions. Said treatment of thenuclei acid preferably comprises use of a reagent selected from thegroup consisting of bisulfite, hydrogen sulfite, disulfite, andcombinations thereof In a preferred embodiment of the invention, theinvention provides a non-naturally occurring modified nucleic acidcomprising a sequence of at least 16 contiguous nucleotide bases inlength of a sequence selected from the group consisting of the bisulfitesequences, in particular from the sequences as defined by SEQ ID NOs: 5,7, 10 to 13 and 18 to 20. In further preferred embodiments of theinvention said nucleic acid is at least 50, 100, 150, 200, 250 or 500base pairs in length of a segment of the nucleic acid sequence disclosedin the bisulfite sequences. Particularly preferred is a nucleic acidmolecule that is identical or complementary to all or a portion of thesequences the bisulfite sequences but not the genomic sequences or othernaturally occurring DNA.

It is preferred that said sequence comprises at least one CpG, TpA orCpA dinucleotide and sequences complementary thereto. The sequences ofthe bisulfite sequences provide non-naturally occurring modifiedversions of the nucleic acid according to the genomic sequences, whereinthe modification of each genomic sequence results in the synthesis of anucleic acid having a sequence that is unique and distinct from saidgenomic sequence as follows. For each sense strand genomic DNA, fourconverted versions are disclosed. A first version wherein “C” isconverted to “T,” but “CpG” remains “CpG” (i.e., corresponds to casewhere, for the genomic sequence, all “C” residues of CpG dinucleotidesequences are methylated and are thus not converted); a second versiondiscloses the complement of the disclosed genomic DNA sequence (i.e.antisense strand), wherein “C” is converted to “T,” but “CpG” remains“CpG” (i.e., corresponds to case where, for all “C” residues of CpGdinucleotide sequences are methylated and are thus not converted). The‘upmethylated’ converted sequences of the genomic sequences correspondto SEQ ID NOs: 4-9 for SEPTIN9 and to SEQ ID NOs: 17 and 18 for RASSF2A.A third chemically converted version of each genomic sequences isprovided, wherein “C” is converted to “T” for all “C” residues,including those of “CpG” dinucleotide sequences (i.e., corresponds tocase where, for the genomic sequences, all “C” residues of CpGdinucleotide sequences are unmethylated); a final chemically convertedversion of each sequence, discloses the complement of the disclosedgenomic DNA sequence (i.e. antisense strand), wherein “C” is convertedto “T” for all “C” residues, including those of “CpG” dinucleotidesequences (i.e., corresponds to case where, for the complement(antisense strand) of each genomic sequence, all “C” residues of CpGdinucleotide sequences are unmethylated). The ‘downmethylated’ convertedsequences of the genomic sequences corresponds to SEQ ID NOs: 10-15 forSEPTIN9 and to SEQ ID NOS: 19 and 20 for RASSF2A.

Significantly, heretofore, the nucleic acid sequences and moleculesaccording the bisulfite sequences were not implicated in or connectedwith the prognosis of a subject having cancer.

In an alternative embodiment, the invention further providesoligonucleotides or oligomers suitable for use in the methods of theinvention for detecting the cytosine methylation state within genomic ortreated (chemically modified) DNA. Said oligonucleotide or oligomernucleic acids provide novel prognostic means. Said oligonucleotide oroligomer comprising a nucleic acid sequence having a length of at leastnine (9) nucleotides which is identical to, hybridizes, under moderatelystringent or stringent conditions (as defined herein above), to atreated nucleic acid sequence according to the bisulfite sequencesand/or sequences complementary thereto, or to a genomic sequenceaccording to the genomic sequences and/or sequences complementarythereto.

Thus, the present invention includes nucleic acid molecules (e.g.,oligonucleotides and peptide nucleic acid (PNA) molecules(PNA-oligomers)) that hybridize under moderately stringent and/orstringent hybridization conditions to all or a portion of the sequencesor to the complements thereof. Particularly preferred is a nucleic acidmolecule that hybridizes under moderately stringent and/or stringenthybridization conditions to all or a portion of the sequences thebisulfite sequences but not the genomic sequences or other human genomicDNA.

The identical or hybridizing portion of the hybridizing nucleic acids istypically at least 9, 16, 20, 25, 30 or 35 nucleotides in length.However, longer molecules have inventive utility, and are thus withinthe scope of the present invention.

Preferably, the hybridizing portion of the inventive hybridizing nucleicacids is at least 95%, or at least 98%, or 100% identical to thesequence, or to a portion thereof, or to the complements thereof.

Hybridizing nucleic acids of the type described herein can be used, forexample, as a primer (e.g., a PCR primer), or a prognostic probe orprimer. Preferably, hybridization of the oligonucleotide probe to anucleic acid sample is performed under stringent conditions and theprobe is 100% identical to the target sequence. Nucleic acid duplex orhybrid stability is expressed as the melting temperature or Tm, which isthe temperature at which a probe dissociates from a target DNA. Thismelting temperature is used to define the required stringencyconditions.

For target sequences that are related and substantially identical to thecorresponding sequence of the genomic sequences (such as allelicvariants and SNPs), rather than identical, it is useful to firstestablish the lowest temperature at which only homologous hybridizationoccurs with a particular concentration of salt (e.g., SSC or SSPE).Then, assuming that 1% mismatching results in a 1° C. decrease in theTm, the temperature of the final wash in the hybridization reaction isreduced accordingly (for example, if sequences having >95% identity withthe probe arc sought, the final wash temperature is decreased by 5° C.).In practice, the change in Tm can be between 0.5° C. and 1.5° C. per 1%mismatch.

Examples of inventive oligonucleotides of length X (in nucleotides), asindicated by polynucleotide positions with reference to the genomic andconverted sequences described herein, include those corresponding tosets (sense and antisense sets) of consecutively overlappingoligonucleotides of length X, where the oligonucleotides within eachconsecutively overlapping set (corresponding to a given X value) aredefined as the finite set of Z oligonucleotides from nucleotidepositions:

n to (n+(X−1));where n=1, 2, 3, . . . (Y−(X−1));where Y equals the length (nucleotides or base pairs) of SEQ ID NOs:1-20;

where X equals the common length (in nucleotides) of eacholigonucleotide in the set (e.g., X=20 for a set of consecutivelyoverlapping 20-mers); and

where the number (Z) of consecutively overlapping oligomers of length Xfor a given SEQ ID NO: of length Y is equal to Y−(X−1).

Preferably, the set is limited to those oligomers that comprise at leastone CpG, TpG or CpA dinucleotide.

Examples of inventive 20-mer oligonucleotides include the oligomersdescribed herein (and the antisense set complementary thereto),indicated by polynucleotide positions with reference to SEQ ID NOs: 1 to20:

1-20, 2-21, 3-22, 4-23, 5-24, etc.

Preferably, the set is limited to those oligomers that comprise at leastone CpG, TpG or CpA dinucleotide.

Likewise, examples of inventive 25-mer oligonucleotides include thefollowing set of xxx oligomers (and the antisense set complementarythereto), indicated by polynucleotide positions with reference to SEQ IDNOs: 1 to 20:

1-25, 2-26, 3-27, 4-28, 5-29, etc.

Preferably, the set is limited to those oligomers that comprise at leastone CpG, TpG or CpA dinucleotide.

The present invention encompasses, for each of the sequences that ismethylated in cancer, but unmethylated in non-cancerous tissue (senseand antisense), multiple consecutively overlapping sets ofoligonucleotides or modified oligonucleotides of length X, where, e.g.,X=9, 10, 17, 20, 22, 23, 25, 27, 30 or 35 nucleotides.

The oligonucleotides or oligomers according to the present inventionconstitute effective tools useful to ascertain genetic and epigeneticparameters of the genomic sequence corresponding to the genomicsequences. Sets of such oligonucleotides or modified oligonucleotidesare those consecutively overlapping sets of oligomers corresponding tosequence that is methylated in cancer, but unmethylated in non-canceroustissue (and to the complements thereof). Preferably, said oligomerscomprise at least one CpG, TpG or CpA dinucleotide.

Particularly preferred oligonucleotides or oligomers according to thepresent invention are those in which the cytosine of the CpGdinucleotide (or of the corresponding converted TpG or CpA dinculcotide)sequences is within the middle third of the oligonucleotide; that is,where the oligonucleotide is, for example, 13 bases in length, the CpG,TpG or CpA dinucleotide is positioned within the fifth to ninthnucleotide from the 5′-end.

The oligonucleotides of the invention can also be modified by chemicallylinking the oligonucleotide to one or more moieties or conjugates toenhance the activity, stability or detection of the oligonucleotide.Such moieties or conjugates include chromophores, fluorophors, lipidssuch as cholesterol, cholic acid, thioether, aliphatic chains,phospholipids, polyamines, polyethylene glycol (PEG), palmityl moieties,and others as disclosed in, for example, U.S. Pat. Nos. 5,514,758,5,565,552, 5,567,810, 5,574,142, 5,585,481, 5,587,371, 5,597,696 and5,958,773. The probes may also exist in the form of a PNA (peptidenucleic acid) which has particularly preferred pairing properties. Thus,the oligonucleotide may include other appended groups such as peptides,and may include hybridization-triggered cleavage agents (Krol et al.,BioTechniques 6: 958-976, 1988) or intercalating agents (Zon, Pharm.Res. 5: 539-549, 1988). To this end, the oligonucleotide may beconjugated to another molecule, e.g., a chromophore, fluorophor,peptide, hybridization-triggered cross-linking agent, transport agent,hybridization-triggered cleavage agent, etc.

The oligonucleotide may also comprise at least one art-recognizedmodified sugar and/or base moiety, or may comprise a modified backboneor non-natural internucleoside linkage.

The oligonucleotides or oligomers according to particular embodiments ofthe present invention are typically used in ‘sets,’ which contain atleast one oligomer for analysis of each of the CpG dinucleotides of agenomic sequence selected from the group consisting the genomicsequences and sequences complementary thereto, or to the correspondingCpG, TpG or CpA dinucleotide within a sequence of the treated nucleicacids according to the bisulfite sequences and sequences complementarythereto. However, it is anticipated that for economic or other factorsit may be preferable to analyse a limited selection of the CpGdinucleotides within said sequences, and the content of the set ofoligonucleotides is altered accordingly.

Therefore, in particular embodiments, the present invention provides aset of at least two (2) (oligonucleotides and/or PNA-oligomers) usefulfor detecting the cytosine methylation state in treated genomic DNA (thebisulfite sequences), or in genomic DNA (the genomic sequences andsequences complementary thereto). These probes enable determination ofthe prognosis of a subject having cancer. The set of oligomers may alsobe used for detecting single nucleotide polymorphisms (SNPs) in treatedgenomic DNA (the bisulfite sequences), or in genomic DNA (the genomicsequences and sequences complementary thereto).

In preferred embodiments, at least one, and more preferably all membersof a set of oligonucleotides is bound to a solid phase.

In further embodiments, the present invention provides a set of at leasttwo (2) oligonucleotides that are used as ‘primer’ oligonucleotides foramplifying DNA sequences of one of sequence that is methylated incancer, but unmethylated in non-cancerous tissue and sequencescomplementary thereto, or segments thereof.

It is anticipated that the oligonucleotides may constitute all or partof an “array” or “DNA chip” (i.e., an arrangement of differentoligonucleotides and/or PNA-oligomers bound to a solid phase). Such anarray of different oligonucleotide- and/or PNA-oligomer sequences can becharacterized, for example, in that it is arranged on the solid phase inthe form of a rectangular or hexagonal lattice. The solid-phase surfacemay be composed of silicon, glass, polystyrene, aluminium, steel, iron,copper, nickel, silver, or gold. Nitrocellulose as well as plastics suchas nylon, which can exist in the form of pellets or also as resinmatrices, may also be used. An overview of the Prior Art in oligomerarray manufacturing can be gathered from a special edition of NatureGenetics (Nature Genetics Supplement, Volume 21, January 1999, and fromthe literature cited therein). Fluorescently labelled probes are oftenused for the scanning of immobilized DNA arrays. The simple attachmentof Cy3 and Cy5 dyes to the 5′-OH of the specific probe are particularlysuitable for fluorescence labels. The detection of the fluorescence ofthe hybridised probes may be carried out, for example, via a confocalmicroscope. Cy3 and Cy5 dyes, besides many others, are commerciallyavailable.

It is also anticipated that the oligonucleotides, or particularsequences thereof, may constitute all or part of an “virtual array”wherein the oligonucleotides, or particular sequences thereof, are used,for example, as ‘specifiers’ as part of, or in combination with adiverse population of unique labeled probes to analyze a complex mixtureof analytes. Such a method, for example is described in US 2003/0013091(U.S. Ser. No. 09/898,743, published 16 Jan. 2003). In such methods,enough labels are generated so that each nucleic acid in the complexmixture (i.e., each analyte) can be uniquely bound by a unique label andthus detected (each label is directly counted, resulting in a digitalread-out of each molecular species in the mixture).

It is particularly preferred that the oligomers according to theinvention are utilised for determining the prognosis of a subject havingcancer.

Kits

Moreover, an additional aspect of the present invention is a kitcomprising: a means for determining methylation of at least one gene orgenomic sequence that is methylated in cancer, but unmethylated innon-cancerous tissue. The means for determining methylation of at leastone gene or genomic sequence that is methylated in cancer, butunmethylated in non-cancerous tissue comprise preferably abisulfite-containing reagent; one or a plurality of oligonucleotidesconsisting whose sequences in each case are identical, arecomplementary, or hybridise under stringent or highly stringentconditions to an at least 9, at least 18, at least 25, or at least 50base long segment of a sequence selected from the bisulfite sequences;and optionally instructions for carrying out and evaluating thedescribed method of methylation analysis. In one embodiment the basesequence of said oligonucleotides comprises at least one CpG, CpA or TpGdinucleotide.

In a further embodiment, said kit may further comprise standard reagentsfor performing a CpG position-specific methylation analysis, whereinsaid analysis comprises one or more of the following techniques:MS-SNuPE, MSP, MethyLight™, HeavyMethyl, COBRA, and nucleic acidsequencing. However, a kit along the lines of the present invention canalso contain only part of the aforementioned components.

In a preferred embodiment the kit may comprise additional bisulfiteconversion reagents selected from the group consisting: DNA denaturationbuffer; sulfonation buffer; DNA recovery reagents or kits (e.g.,precipitation, ultrafiltration, affinity column); desulfonation buffer;and DNA recovery components.

In a further alternative embodiment, the kit may contain, packaged inseparate containers, a polymerase and a reaction buffer optimised forprimer extension mediated by the polymerase, such as PCR. In anotherembodiment of the invention the kit further comprising means forobtaining and/or storing a biological sample of the subject. Preferredis a kit, which further comprises a container suitable for containingthe means for determining methylation of at least one gene or genomicsequence that is methylated in cancer, but unmethylated in non-canceroustissue in the biological sample of the subject, and most preferablyfurther comprises instructions for use and interpretation of the kitresults. In a preferred embodiment the kit comprises: (a) a bisulfitereagent; (b) a container suitable for containing the said bisulfitereagent and the biological sample of the subject; (c) at least one setof primer oligonucleotides containing two oligonucleotides whosesequences in each case are identical, are complementary, or hybridiseunder stringent or highly stringent conditions to an at least 9 or morepreferably 18 base long segment of a sequence selected from thebisulfite sequences; and optionally (d) instructions for use andinterpretation of the kit results. In an alternative preferredembodiment the kit comprises: (a) a bisulfite reagent; (b) a containersuitable for containing the said bisulfite reagent and the biologicalsample of the subject; (c) at least one oligonucleotides and/orPNA-oligomer having a length of at least 9 or 16 nucleotides which isidentical to or hybridises to a pre-treated nucleic acid sequenceaccording to one of the bisulfite sequences and sequences complementarythereto; and optionally (d) instructions for use and interpretation ofthe kit results.

In an alternative embodiment the kit comprises: (a) a bisulfite reagent;(b) a container suitable for containing the said bisulfite reagent andthe biological sample of the subject; (c) at least one set of primeroligonucleotides containing two oligonucleotides whose sequences in eachcase are identical, are complementary, or hybridise under stringent orhighly stringent conditions to an at least 9 or more preferably 18 baselong segment of a sequence selected from the bisulfite sequences; (d) atleast one oligonucleotides and/or PNA-oligomer having a length of atleast 9 or 16 nucleotides which is identical to or hybridises to apre-treated nucleic acid sequence according to one of the bisulfitesequences and sequences complementary thereto; and optionally (e)instructions for use and interpretation of the kit results.

The kit may also contain other components such as buffers or solutionssuitable for blocking, washing or coating, packaged in a separatecontainer.

Another aspect of the invention relates to a kit for use in determiningthe prognosis of a subject having cancer, said kit comprising: a meansfor measuring the level of transcription of at least one gene or genomicsequence that is methylated in cancer, but unmethylated in non-canceroustissue and a means for determining methylation of at least one gene orgenomic sequence that is methylated in cancer, but unmethylated innon-cancerous tissue.

Typical reagents (e.g., as might be found in a typical COBRA™-based kit)for COBRA™ analysis may include, but are not limited to: PCR primers forat least one gene or genomic sequence that is methylated in cancer, butunmethylated in non-cancerous tissue restriction enzyme and appropriatebuffer; gene-hybridization oligo; control hybridization oligo; kinaselabeling kit for oligo probe; and labeled nucleotides. Typical reagents(e.g., as might be found in a typical MethyLight™-based kit) forMethyLight™ analysis may include, but are not limited to: PCR primersfor the bisulfite converted sequence of at least one gene or genomicsequence that is methylated in cancer, but unmethylated in non-canceroustissue bisulfite specific probes (e.g. TaqMan™ or LightCycler™);optimized PCR buffers and deoxynucleotides; and Taq polymerase.

Typical reagents (e.g., as might be found in a typical Ms-SNuPE™-basedkit) for Ms-SNuPE™ analysis may include, but are not limited to: PCRprimers for specific gene (or bisulfite treated DNA sequence or CpGisland); optimized PCR buffers and deoxynucleotides; gel extraction kit;positive control primers; Ms-SNuPE™ primers for the bisulfite convertedsequence of at least one gene or genomic sequence that is methylated incancer, but unmethylated in non-cancerous tissue reaction buffer (forthe Ms-SNuPE reaction); and labelled nucleotides.

Typical reagents (e.g., as might be found in a typical MSP-based kit)for MSP analysis may include, but are not limited to: methylated andunmethylated PCR primers for the bisulfite converted sequence of atleast one gene or genomic sequence that is methylated in cancer, butunmethylated in non-cancerous tissue, optimized PCR buffers anddeoxynucleotides, and specific probes.

Moreover, an additional aspect of the present invention is analternative kit comprising a means for determining at least one gene orgenomic sequence that is methylated in cancer, but unmethylated innon-cancerous tissue methylation, wherein said means comprise preferablyat least one methylation specific restriction enzyme; one or a pluralityof primer oligonucleotides (preferably one or a plurality of primerpairs) suitable for the amplification of a sequence comprising at leastone CpG dinucleotide of a sequence selected from the genomic sequences;and optionally instructions for carrying out and evaluating thedescribed method of methylation analysis. In one embodiment the basesequence of said oligonucleotides are identical, are complementary, orhybridise under stringent or highly stringent conditions to an at least18 base long segment of a sequence selected from the genomic sequences.

In a further embodiment said kit may comprise one or a plurality ofoligonucleotide probes for the analysis of the digest fragments,preferably said oligonucleotides are identical, are complementary, orhybridise under stringent or highly stringent conditions to an at least16 base long segment of a sequence selected from the genomic sequences.

In a preferred embodiment the kit may comprise additional reagentsselected from the group consisting: buffer (e.g. restriction enzyme,PCR, storage or washing buffers); DNA recovery reagents or kits (e.g.,precipitation, ultrafiltration, affinity column) and DNA recoverycomponents.

In a further alternative embodiment, the kit may contain, packaged inseparate containers, a polymerase and a reaction buffer optimised forprimer extension mediated by the polymerase, such as PCR. In anotherembodiment of the invention the kit further comprising means forobtaining and/or storing a biological sample of the subject. In apreferred embodiment the kit comprises: (a) a methylation sensitiverestriction enzyme reagent; (b) a container suitable for containing thesaid reagent and the biological sample of the subject; (c) at least oneset of oligonucleotides one or a plurality of nucleic acids or peptidenucleic acids which are identical, are complementary, or hybridise understringent or highly stringent conditions to an at least 9 base longsegment of a sequence selected from the genomic sequences; andoptionally (d) instructions for use and interpretation of the kitresults. In an alternative preferred embodiment the kit comprises: (a) amethylation sensitive restriction enzyme reagent; (b) a containersuitable for containing the said reagent and the biological sample ofthe subject; (c) at least one set of primer oligonucleotides suitablefor the amplification of a sequence comprising at least one CpGdinucleotide of a sequence selected from the genomic sequences; andoptionally (d) instructions for use and interpretation of the kitresults.

In an alternative embodiment the kit comprises: (a) a methylationsensitive restriction enzyme reagent; (b) a container suitable forcontaining the said reagent and the biological sample of the subject;(c) at least one set of primer oligonucleotides suitable for theamplification of a sequence comprising at least one CpG dinucleotide ofa sequence selected from the genomic sequences ; (d) at least one set ofoligonucleotides one or a plurality of nucleic acids or peptide nucleicacids which are identical , are complementary, or hybridise understringent or highly stringent conditions to an at least 9 base longsegment of a sequence selected from the genomic sequences and optionally(e) instructions for use and interpretation of the kit results.

The kit may also contain other components such as buffers or solutionssuitable for blocking, washing or coating, packaged in a separatecontainer.

The invention further relates to a kit for use in determining theprognosis of a subject having cancer, in a subject by means ofmethylation-sensitive restriction enzyme analysis. Said kit comprises acontainer and a DNA microarray component. Said DNA microarray componentbeing a surface upon which a plurality of oligonucleotides areimmobilized at designated positions and wherein the oligonucleotidecomprises at least one CpG methylation site. At least one of saidoligonucleotides is specific for at least one gene or genomic sequenceselected from the group consisting of the genes and comprises a sequenceof at least 15 base pairs in length but no more than 200 bp of asequence according to one of the genomic sequences . Preferably saidsequence is at least 15 base pairs in length but no more than 80 bp of asequence according to one of the genomic sequences . It is furtherpreferred that said sequence is at least 20 base pairs in length but nomore than 30 bp of a sequence according to one of the genomic sequences.Said test kit preferably further comprises a restriction enzymecomponent comprising one or a plurality of methylation-sensitiverestriction enzymes.

In a further embodiment said test kit is further characterized in thatit comprises at least one methylation-specific restriction enzyme, andwherein the oligonucleotides comprise a restriction site of said atleast one methylation specific restriction enzymes.

The kit may further comprise one or several of the following components,which are known in the art for DNA enrichment: a protein component, saidprotein binding selectively to methylated DNA; a triplex-forming nucleicacid component, one or a plurality of linkers, optionally in a suitablesolution; substances or solutions for performing a ligation e.g.ligases, buffers; substances or solutions for performing a columnchromatography; substances or solutions for performing an immunologybased enrichment (e.g. immunoprecipitation); substances or solutions forperforming a nucleic acid amplification e.g. PCR; a dye or several dyes,if applicable with a coupling reagent, if applicable in a solution;substances or solutions for performing a hybridization; and/orsubstances or solutions for performing a washing step.

The described invention further provides a composition of matter usefulfor determining the prognosis of a subject having cancer . Saidcomposition comprising at least one nucleic acid 18 base pairs in lengthof a segment of the nucleic acid sequence disclosed in the bisulfitesequences, and one or more substances taken from the group comprising :1-5 mM Magnesium Chloride, 100-500 μM dNTP, 0.5-5 units of taqpolymerase, bovine serum albumen, an oligomer in particular anoligonucleotide or peptide nucleic acid (PNA)-oligomer, said oligomercomprising in each case at least one base sequence having a length of atleast 9 nucleotides which is complementary to, or hybridizes undermoderately stringent or stringent conditions to a pretreated genomic DNAaccording to one of the bisulfite sequences and sequences complementarythereto. It is preferred that said composition of matter comprises abuffer solution appropriate for the stabilization of said nucleic acidin an aqueous solution and enabling polymerase based reactions withinsaid solution. Suitable buffers arc known in the art and commerciallyavailable.

The present invention also relates to the use of a kit or anoligonucleotide as defined above for determining the prognosis of acancer subject, determining medical treatment for a cancer subject,determining if a tumor from a cancer subject indicates that the tumor isaggressive or has metastatic potential or indicates a reduced survivaltime for the subject, detecting an aggressive form of cancer in asubject, selecting a cancer subject for cancer treatment, or determiningtumor load or cancer burden in a subject comprising of a cancer subject.

In further preferred embodiments of the invention said at least onenucleic acid is at least 50, 100, 150, 200, 250 or 500 base pairs inlength of a segment of the nucleic acid sequence disclosed in thebisulfite sequences.

TABLE 1 Genomic sequences and treated variants thereof according to theinvention Methylated Methylated Unmethylated Unmethylated Ensemblbisulfite bisulfite bisulfite bisulfite SEQ Ensembl datanbase* convertedconverted converted converted ID database* genomic Associated genesequence sequence sequence sequence NO: location location transcript(s)*(sense) (antisense) (sense) (antisense) 1 AC068594.15.1.168501 17 Septin9 & 4 5 10  11] 150580 to 151086 (+) to 72789082 to Q9HC74AC111170.11.1.158988 73008258 (+) 137268 to 138151 (+) 2AC068594.15.1.168501 17 Septin 9 6 7 12 13 150580 to 151255 (+) 72789082to 72789757 (+) 3 AC111182.20.1.171898 17 Q9HC74 8 9 14 15 127830 to129168 (+) 72881422 to 72882760 (+) 16 RASSF2A 17 18 19 20

EXAMPLE 1

Levels of Septin9 and methylated RASSF2A as examples of genes or genomicsequences that are methylated in cancer tissue but unmethylated innon-cancerous tissue were investigated in matched plasma samples fromCRC patients pre- and post- surgical resection.

Levels of Septin9 and RASSF2A were determined by the triplex assaymethod measuring Septin9, methylated RASSF2A, and HB14 genes. Similarassays can be run in singleplex, duplex, triplex, quadriplex, ormultiplex format.

Method for measuring methylation or the methylation status of genes andgenomic sequences are known in the art. See, for example, U.S. Pat. No.7,229,759, or European Patent No: EP 1370691, both of which areincorporated herein for reference to these methylation assay anddetection methods. The methylation or the methylation status of genesand genomic sequences herein was measured. The plasma DNA was bisulfiteconverted and the level of methylated DNA at positions on the genomicsequences was detected in a triplex assay.

Invitrogen magnetic racks (DynaMag-15 and DynaMag-2) were used. Wash Awas prepared by adding 45 ml Ethanol (Merck, A000920; 99.8%) to the EpiproColonUS Wash A Concentrate. Wash B was prepared by adding 28 mlEthanol (Merck, A000920; 99.8%) to the Epi proColonUS Wash BConcentrate. To a labeled 15 ml Falcon tube, 3,5 ml of blood plasma wascombined with 3.5 ml Lysis-Binding buffer, mixed by vortexing, andincubated at room temperature for 10 min. To this lysis reaction 90 μIMagnetic Beads(Dynabeads MyOne SILANE, freshly suspended by vortexingfor 30 seconds) and 2,5 ml Ethanol was added to a total volume will be˜10 ml. The tube was mix by inverting by hand 5-6 times and incubatedwith rotating shaker (Rotator) for 45 min at room temperature. The firstwash was performed by placing 15 ml tubes into magnetic rack for atleast 5 min after which the buffer was discarded and the tubes weretransferred into a non-magnetic rack. 1500 μl Wash A (as described aboveLysis/Binding Buffer+Ethanol f. d. Molekularbiologie) was added and thebeads were resuspended by vortexing for 10 sec. The bead suspension wastransferred into a labeled 2 ml tube with transfer pipette. 2 mlSafeLock tubes only to ensure safe closure during 80° C. incubation wereused for centrifugation. The transfer pipette was placed back into the15 ml tube for at least 2 min to collect remaining beads. And placedinto the 2 ml tube with same transfer pipette. The 2 ml tubes wereplaced on the magnetic rack for 2 min after which as much wash buffer aspossible was pipette off while taking care not to remove beads. Thetubes were placed in a centrifuge and spun for 10 sec at 1000 rcf tocollect beads at the bottom, the placed on the magnetic rack for 2 minand remove residual Buffer.

Plasma DNA was eluted by adding 100 μl Elution buffer (10 mM Tris pH8.0) to each tube and the beads were resuspended by vortex 10 sec,making sure that the pellet was been resuspended completely, thenincubated at 80° C. for 15 min in a thermal shaker at 1000 rpm. Thetubes were pulse spun to remove drops from the lid. And placed on themagnetic rack for 2 min. The complete eluate (circa 100 μl) wastransferred into prelabeled 2.0 ml tubes

The plasma DNA was bisulfite converted by adding the following reagentsto the eluate: 150 μl Bisulfite Solution (ABS, Ammonium bisulfitesolution, use unopened tubes only, discard used tubes) and 25 μlProtection Buffer (contains THFA) (5 g Trolox+40 ml THFA). The tubeswere closed and vortexed for 10 seconds to mix thoroughly. The tubeswere pulse spun to prevent liquid on the lid, then placed into a thermalblock or shaker and incubated for 45 min at 80° C. without shaking. Thetubes were pulse spun to remove drops from the lid, then the beads wereresuspended by vortexing for 10 seconds, making sure that all beads arethoroughly suspended. The following components were added into eachbisulfite reaction in order: 1000 μl Wash A and 20 μl Magnetic Beads(Dynabeads MyOne SILANE) to a total volume of 300 ul and mixed carefullyby vortexing, after which they were incubated at thermal shaker andshake at 1000 rpm for 45 min at room temperature. The tubes were pulsespun tube to remove drops from the lid and placed on the magnetic rackfor 2 min to capture the particles. Then a fresh pipette was used toremove as much liquid as possible without touching the capturedparticles. The tubes were removed from the magnetic rack for the washingprocedure. 800 μl Wash A was added and the beads were rinsed from thewall, then resuspended by vortexing, pulse spun tube to remove dropsfrom the lid, and placed on the magnetic rack for 2 min. Using a freshpipet as much liquid was removed as possible, without touching thecaptured particles. The tubes were taken off the magnetic rack for thewashing procedure. 800 μl Wash B was added, and the beads were rinsedfrom the wall, resuspended by vortexing and pulse spun tube to removedrops from the lid, after which they were placed on the magnetic rackfor 2 min. Using a fresh pipet as much liquid was removed as possible,without touching the captured particles. The tubes were taken off themagnetic rack for the washing procedure. 400 μl Wash B was added and thebeads were rinsed from the wall, resuspended by vortexing, pulse spun toremove drops from the lid and placed on the magnetic rack for 2 min.Using a fresh pipet as much liquid was removed as possible, withouttouching the captured particles. The tubes were briefly spun to collectremaining drops to the bottom, placed on the magnetic rack for 2 minthen the residual liquid was removed with a pipette. The pellet wasallowed to dry for 10 min at room temperature with open tubes on themagnet.

The tubes were transferred into a non-magnetic rack and 55 μl Elutionbuffer (10 mM Tris pH 8.0) to was added to each tube. The beads werethen resuspended by vortex 20 sec.min., after which the tubes wereincubated at 80° C. for 5 min in a thermal shaker at 1000 rpm, thenvortexed again for 10 sec., briefly spun down to collect all liquid downto the bottom. The tubes were placed on the magnetic rack for 2 min. andthe complete eluate was transferred into a 96 well PCR plate (orprelabeled 0.5 ml tubes).

Sequences of probes and primers for triplex assay performed are shown inTable 2

TABLE 2 SEQ Func- ID Oligo Name Gene tion Sequence NO: 10307-92 RASSF2APrimer ctaaaacctcaacctaac 21 10307-94 RASSF2A Primergatttagagttgaatgtaaagtaa 22 10307-9B1 RASSF2A Blockercctaacatcttctctcaccccaaacaaaaca 23 10307-9taq2 RASSF2A Probetaccgtaaacgaccccga 24 17378-109 Septin 9 Primer gttgtttattagttattatgt 25Sept9 R 102 Septin 9 Primer aaataatcccatccaacta 26 Septin9 blockerSeptin 9 Blocker gttattatgttggattttgtggttaatgtgtag 27 17378-10taq4-TAMSeptin 9 Probe ttaaccgcgaaatccgac 28 HB14.F.2short HB 14 Primergtgatggaggaggtttagtaagtt 29 HB14.R.2short HB 14 Primerccaataaaacctactcctcccttaa 30 HB14.taq1-BNM5 HB 14 Probeaccaccacccaacacacaataacaaacaca 31

Septin9 Genomic Sequence

(SEQ ID NO: 32) Ctgcccaccagccatcatgtcggaccccgcggtcaacgcgcagctggatgggatcattt Septin9 bisulfite converted genomic sequence: (SEQ ID NO: 33)Ttgtttattagttattatgtcggatttcgcggttaacgcgtagttggat gggattatttRASSF2A genomic sequence: (SEQ ID NO: 34)Acttagagctgaatgcaaagtaagcgctcgaaatgcagaagtagccggggccgcccacggcacctgcctcgctcggggcgagagaagacgccaggctg aggtcccagRASSF2A bisulfite converted genomic sequence: (SEQ ID NO: 35)atttagagttgaatgtaaagtaagcgttcgaaatgtagaagtagtcggggtcgtttacggtatttgtttcgttcggggcgagagaagacgttaggttg aggttttag

TABLE 3 SEPTIN9 SEPTIN9 RASSF2A RASSF2A Number positive positivepositive positive of BEFORE AFTER BEFORE AFTER CRC Stage Patientssurgery surgery surgery surgery Stage I 4 1/4 0/4 1/4 0/4 Stage II 9 9/94/9 6/9 5/9 Stage III 4 4/4 2/4 4/4 2/4 Stage IV 2 2/2 2/2 2/2 2/2

The results show that Patients with stage I and II cancers tend to loseSeptin9 and RASF2A signal after the surgery, patients with stage IIItend to retain the Septin9 and RASF2A signal. The 2 stage IV CRCpatients, who already have metastatic disease “retain” the Septin9 andRASF2A signal after surgery. This indicates that the metastasies can bedetected by Septin9 and RASF2A even if the primary tumor was resected.The results are depicted in FIGS. 1-12.

FIGS. 14 and 15 display the discussed results of Septin9 and RASSF2A ina quantitative matter. The values are indicated in pg methylatedSeptin9/RASSF2A DNA per ml plasma.

Method for Prognosis of CRC Patients After Curative Resection of thePrimary Tumor

The detection of Septin9 can be done by several state of the arttechnologies that can detect the DNA methylation in blood/plasma. Aqualitative, semiquantitave or/and quantitative analysis of mSeptin9 ispossible and is highly connected to the intended use and thepatient/tumor population of interest.

Sample Determination of Analysis For Qualitative Analysis Stage I CRCTumor Patient

Before surgery Septin9 signal positiveAfter surgery Septin9 signal negative=good prognosisAfter surgery Septin9 signal positive=bad prognosis (risk of metastasis)Good prognosis shall mean in preferred embodiments of the invention thatthe individual who underwent surgery is monitored i.e. one or more testsfor the re-occurance of cancer are repeated in time intervals. In aparticular preferred embodiment such a test is the detection ofmethylated Septin 9 DNA as it is disclosed herein, in US 2006-0286576,or WO 2006/113466.Before surgery RASSF2A signal positiveAfter surgery RASSF2A signal negative=good prognosisAfter surgery RASSF2A signal positive=bad prognosis (risk of metastasis)Good prognosis shall mean in preferred embodiments of the invention thatthe individual who underwent surgery is monitored i.e. one or more testsfor the re-occurance of cancer are repeated in time intervals.

For Semiquantitaive Analysis Stage I, II and III CRC Tumor Patients

Before surgery Septin9 signal positive (1 of 3 replicatemeasurements)=presence of tumorAfter surgery Septin9 signal negative=good prognosis (3 of 3 replicatemeasurements)After surgery Septin9 signal 1 of 3 positive=low riskAfter surgery Septin9 signal 2 of 3 positive=medium riskAfter surgery Septin9 signal 3 of 3 positive=high risk

Stage I, II and III CRC Tumor Patients

Before surgery RASSF2A signal positive (1 of 3 replicatemeasurements)=presence of tumorAfter surgery RASSF2A signal negative=good prognosis (3 of 3 replicatemeasurements)After surgery RASSF2A signal 1 of 3 positive=low riskAfter surgery RASSF2A signal 2 of 3 positive=medium riskAfter surgery RASSF2A signal 3 of 3 positive=high risk

For Quantitative Analysis Stage II and III and (IV) CRC Tumor Patients

Detection of magnitude of Septin9 before and after surgery e.g. by usingan internal standard.

Stage I, II and III CRC Tumor Patients

Before surgery Septin9 above 3 pg/ml plasma=presence of tumorAfter surgery Septin9 signal negative=good prognosis (0 pg/ml Septin9)After surgery Septin9>0 to 3 pg/ml plasma=low riskAfter surgery Septin9 from 3 to 30 pg/ml plasma=medium riskAfter surgery Septin9 above 30 pg/ml plasma=high riskDetection of magnitude of RASSF2A before and after surgery e.g. by usingan internal standard.

Stage I, II and III CRC Tumor Patients

Before surgery RASSF2A above 3 pg/ml plasma=presence of tumorAfter surgery RASSF2A signal negative=good prognosis (0 pg/ml plasmaRASSF2A)After surgery RASSF2A->0 to 3 pg/ml plasma=low riskAfter surgery RASSF2A from 3 to 30 pg/ml plasma=medium riskAfter surgery RASSF2A above 30 pg/ml plasma RASSF2A=high risk

1-43. (canceled)
 44. A method for determining methylation of SEPTIN-9(SEQ ID NO: 1) genomic colon or colorectal tumor cell DNA of a humansubject colorectal cancer following removal of a primary colon orcolorectal tumor but prior to further treatment, wherein the colorectalcancer is staged as Stage I, II or III according to theTumor-Node-Metastasis (TNM) staging method and the removal of a primarycolon or colorectal tumor staged as stage III includes nearby lymphnodes, comprising: detecting the presence of, or measuring the level of,methylated genomic SEPTIN-9 DNA in a stool, blood, serum or plasmasample obtained from the human subject following the removal of theprimary colon or colorectal tumor, wherein the methylated genomicSEPTIN-9 DNA is from colon or colorectal tumor cells, therebydetermining methylation of the SEPTIN-9 genomic colon or colorectaltumor cell DNA of the human subject after removal of the primary colonor colorectal tumor.
 45. The method according to claim 44, wherein themethylation of the SEPTIN-9 genomic colon or colorectal tumor cell DNAis measured quantitatively, or quantitatively in part, qualitatively,and/or qualitatively in part, quantitatively in part and qualitativelyin part, or semi quantitatively.
 46. The method according to claim 44,wherein the methylation of the SEPTIN-9 genomic colon or colorectaltumor cell DNA is detected or measured at least at one cytosine selectedfrom the group consisting of positions 21, 28, 30, 37 and 39 of SEQ IDNO:
 32. 47. The method of claim 44, wherein the colorectal cancer isstaged as Stage I according to the TNM staging method when the primarytumor is removed by surgery or resection.
 48. The method of claim 47,wherein the surgery or resection includes nearby lymph nodes.
 49. Themethod of claim 44, wherein the colorectal cancer is staged as Stage IIaccording to the TNM staging method when the primary tumor is removed bysurgery or resection.
 50. The method of claim 49, wherein the surgery orresection includes nearby lymph nodes.
 51. The method of claim 44,wherein the colorectal cancer is staged as Stage III according to theTNM staging method when the primary tumor is removed by surgery orresection.
 52. The method of claim 44, wherein detecting the presence ofor measuring the level of methylated genomic SEPTIN-9 DNA furthercomprises: contacting the genomic DNA from the stool, blood, serum, orplasma sample of the human subject with a reagent selected from thegroup consisting of bisulfate, hydrogen sulfite, disulfite, andcombinations thereof; and detecting the presence of or measuring thelevel of a nucleic acid sequence comprising SEQ ID NO: 33 in the stool,blood, serum, or plasma sample of the human subject.
 53. The method ofclaim 44, wherein the detection step is performed using quantitativePCR.
 54. The method of claim 53, wherein the detection step is performedwithout confirmation.
 55. The method of claim 53, wherein the detectionstep is performed without confirmation using sequencing.
 56. The methodaccording to claim 44, wherein detecting the presence of or measuringthe level of methylated genomic SEPTIN-9 DNA uses a primer pair in whichone of the primers hybridizes under stringent conditions to at least 9nucleotides of the nucleic acid sequence of SEQ ID NO:26 or a complementthereof, and the other primer of the primer pair hybridizes understringent conditions to at least 9 nucleotides of the nucleic acidsequence of SEQ ID NO:25 or a complement thereof.
 57. The method ofclaim 56, wherein one of the primers of the primer pair comprises thenucleic acid sequence of SEQ ID NO: 26, or a complement thereof, and theother primer of the primer pair hybridizes under stringent conditions toat least 9 nucleotides of the nucleic acid sequence of SEQ ID NO:25 or acomplement thereof.
 58. The method of claim 56, further comprising usinga blocker that hybridizes under stringent conditions to at least 9nucleotides of the nucleic acid sequence of SEQ ID NO:
 27. 59. Themethod of claim 58, further comprising using a probe that hybridizesunder stringent conditions to at least 9 nucleotides of the nucleic acidsequence of SEQ ID NO:
 28. 60. The method of claim 56, wherein at leastone primer of the primer pair comprises SEQ ID NO: 26, or a complementthereof.
 61. The method of claim 56, wherein at least one primer of theprimer pair comprises SEQ ID NO: 25, or a complement thereof.
 62. Themethod of claim 56, wherein determining methylation of the SEPTIN-9genomic colon or colorectal tumor cell DNA further comprises usingprimers consisting of SEQ ID NOs: 25-26, a blocker consisting of SEQ IDNO: 27, and a probe consisting of SEQ ID NO: 28.